In the past, Hamato Yoshi was an orphan, living in the streets of Japan with his girlfriend Tang Shen, in the 1970s. When Yoshi returned a 5-yen coin dropped by the Ancient One, he took them in for their honesty, raised them as his son and daughter. Shen loved Yoshi more. During this time, Shen found a rat in the house and kept him as a pet. One night, Yoshi, Shen, and the Ancient One saw a lone man being attacked by four thugs. Yoshi decided to help the man and fought off the thugs. The man was actually the Utrom Mortu and the thugs were members of the Purple Dragons. Impressed by their skill and already having his secret revealed due to battle damage, Mortu offered to make the Guardian. The Ancient One will welcome Mortu and became a good friends.
As a Guardian, Yoshi seemed to advance continually, while the former guardian Oroku Saki never seemed to get anywhere. In anger, he became the Shredder and betrayed the Utroms. Yoshi and the Utroms managed to escape with their lives. When Yoshi returned to the Ancient One, the ninjas kidnapped Tang Shen and Yoshi was unable to save her in time. In anger, despite counsel from his master, Yoshi stormed the Foot's citadel and rescued Shen from the Shredder.
With their Tokyo base discovered, the Utrom set up a new base in New York under the name T.C.R.I. Yoshi and Shen went with them, taking their rat with them. Shen named the rat Splinter to remind Yoshi of the splinters of anger and hate that drove him to defeat the Shredder. At some point Yoshi married Shen and gave birth to their baby son Shouta. However, the Shredder followed Yoshi and Shen and tried to force him to reveal the location of the Utroms. Yoshi and Shen resisted and were killed. Splinter escaped to the sewers, where he mutated along with the Turtles and Shouta was rescued and then adopted by Mortu and other Utroms.
In the present, Splinter and the Turtles are now became a sentients, contained in a research facility called Genius Incorporated, owned by Baxter Stockman, an African American scientist who has secretly ailied himself with Krang, a ruthless and corrupt Utrom from Dimension X. But the Foot Clan still lives, and they break into Genius Incorporated one night and steal some radioactive chemicals which, after a long chase, end up mutating Splinter and the turtles into humanoid creatures.
Splinter, remembering his previous life, decides to train his sons in Ninjutsu.
- Teenage Mutant Ninja Turtles - A team of anthropomorphic freshwater turtles who are the protagonists of the series:
- Leonardo - "Leo" is a hardworking ninjutsu student, learning how to be an effective leader for his three often unruly brothers. He wears a blue mask and fights with two katana swords called Niten Ryu. He also has a crush on Bunny which his brothers all tease him about.
- Donatello - "Donnie" is in charge of the design and manufacture of all of the tools and weapons in the Turtles' arsenal, and he is also a gifted hacker. He wears a purple mask and fights with a rokushakubo which converts to a naginata.
- Raphael - "Raph" often lets his impulsiveness get in the way of rational thinking. He wears a red mask and fights with two sai.
- Michelangelo - "Mikey" loves video games, skateboarding, pranks and pizza. He wears an orange mask and fights with two nunchakuwhich convert into kusarigama. Though he possesses a child-like innocence, Mikey always brings his best game to a fight.
- Splinter - Splinter is the mutant rat leader of the entire family, and serves as a father figure for the Turtles. He once was a pet for a renowned martial artist, Hamato Yoshi in Japan. It was from watching Yoshi that Splinter first learned the art of ninjitsu which he then passes on to the Turtles. Splinter often acts as much as a spiritual guide and counselor to the team as he does their teacher. While he does not wield a traditional Japanese weapon, Splinter carries a cane which he can utilize as a weapon (like a bo staff) with fierce proficiency. Unlike the previous animated series, the Turtles often refer to Splinter as their father, rather than their master.
- April O'Neil - She is the Turtles' closest human ally and "big sister". When they first meet April is working under Baxter Stockman as a technical assistant. April is tech savvy on a level close to Donatello, making them fast friends once they become allies. April often assists the Turtles with recon on various missions. She later offers her apartment to the Turtles as a temporary home. April's apartment is located above the Second Time Around antique store which she owns and operates. April serves as the Turtles first true glimpse into human interaction and helps them learn about the outside world. She later becomes romantically involved with Casey Jones.
- Casey Jones - He is a confidant and friend to the Turtle family. He operates as a vigilante, at first on his own, and then later as a member of the Turtles' extended crew. While at first being more closely aligned with Raphael, Casey quickly becomes part of the family and valuable fighter. When in his full get-up he wears a hockey mask, and utilizes a hockey stick and other sports paraphernalia as his weapons. He takes to April quite quickly, and although she dislikes him initially the two become romantically involved.
- Bunny - Bunny is a mutated white rabbit who was taken in by the Utroms when they discovered that their mutagen (the same that transformed the Turtles) made her sentient. She also wears a sleeveless pink dress held up by a white-trimmed halter strap with matching trim on the skirt and lined with a white slip, along with white go-go shoes.
- Leatherhead - Leatherhead is a mutated green alligator who was taken in by the Utroms when they discovered that their mutagen (the same that transformed the Turtles) made him sentient. Leatherhead lived with the Utroms peacefully until Krang's attack forced him into hiding. Some time later Baxter Stockman fooled him into aiding him with a scheme to restore his body. Ultimately, Leatherhead learned that Stockman had ties to the Shredder and wound up fighting on the side of good. As the series progresses he becomes a semi-regular member of the Turtles extended family.
- Foot Clan - An evil ninja organization that led by the Shredder.
- Shredder - A major antagonist of the series and leader of the Foot Clan.
- Karai - A rebellious teen member of the Foot Clan and an expert kunoichi who stirs up trouble for the Turtles. She is an adoptive daughter of the Shredder.
- Erik Wackwood/Armaggon - Erik Wackwood is a famous martial artist and secretly a member of the Foot Clan. He is ordered to New York with Maurice to find Splinter. As Wackwood was previously bitten by a great white shark, the mutagen transforms him into Armaggon, a shark-like mutant with large hands and legs.
- Maurice/Buttercup - Maurice is a fat English street thug who was in a prison. He learned how to make weapons in prison until Shredder freed him and brought him into the Foot Clan. Shredder orders Maurice to assist Erik Wackwood in finding Splinter. Maurice hates what he calls "freaks", which are basically any being other than a human, even to point of hating and being ashamed of working for Shredder. As Maurice was previously petted his pet bulldog, Jack, the mutagen transforms him into Buttercup, a hunchbacked, dog-like mutant with a muscular arms, acute senses, and super-strength.
- Larry, Mack, and Horace/Triple Threat - Larry, Mack, and Horace are the three street wrestlers who were originally experimented by Krang and merged into a three-headed Triple Threat. His three heads are a head of African American wrestler, Mack with a mustache on the far right side of his body, a head of Brooklyn wrestler, Larry with a mohawk and goatee in the middle, and a head of Australian wrestler, Horace with bald head on the far left side. Shredder brought them (or him) into the Foot Clan.
- Bob/El Gorila - A Mexican thug working for Krang and his Rock Soldiers as their gateway driver. The Turtles interrogate him and find out where General Krang and the Rock Soldiers are holding Bunny. During their mission to rescue Bunny, the Turtles crash a truck full of mutagen into the Genius Incorporated base. Bob is exposed to the the mutagen after he shakes hands with the gorilla. He transforms into a spike-backed, gorilla-like monster, El Gorila. The Turtles fight El Gorila. Leonardo lures him into a sewers where El Gorila falls down. After the Turtles leave, El Gorila is now furious that he has been transformed into a gorilla monster, the one thing he hates most in the world, even more than the Shredder. Consumed with hatred, he joins the Shredder to help him destroy the Turtles.
- Bebop and Rocksteady - Two punks working for Krang and his Rock Soldiers as their guards. The Turtles interrogate them and find out where General Krang and the Rock Soldiers are holding April. During their mission to rescue April, the Turtles crash a truck full of mutagen into Krang's base. They are exposed to mutagen that turned by Bebop into a mutant warthog and Rocksteady into a mutant rhinoceros. Later, they join the Shredder to help him destroy the Turtles.
- Foot Ninjas - The foot soldiers of the Foot Clan.
- Krang's Army - An evil organization that led by General Krang.
- General Krang - The most ruthless and corrupt criminal the universe has known. Krang dreams of galactic domination, and wants vengeance against the Utroms for imprisoning him and denying him his dreams by building an army and conquering their homeworld. In seemingly separate wars with the Triceratons and the Foot Clan, Krang had bankrolled Baxter Stockman to develop a mutagen that would strengthen his soldiers.
- Captain Traag - A second in command of the Rock Soldiers from Dimension X. He is loyal to Krang.
- Sergeant Granitor - One of Captain Traag's underlings.
- Rock Soldiers - A rock monsters of Krang's army.
- Genius Incorporated - A research lab owned by Baxter Stockman who has secretly ailied himself with Krang.
- Baxter Stockman - An owner of the Genius Incorporated Laboratories.
- Mousers - Mousers are a small robots that were created by Baxter Stockman to steal on his behalf.
- Dr. Chapman - A Native American scientist who is Baxter Stockman's assistant.
- Jack/Old Humb - Jack was a one-eyed street thug who along with McBison and Black Hare became Stockman's right hand men following his mutation into Old Humb, a hamster-like mutant.
- Muck/McBison - Muck was a Russian thug who along with Old Humb and Black Hare became Stockman's right hand men following his mutation into McBison, a muscular, bison-like mutant.
- Harold/Black Hare - Harold was a Spainish thug who along with Old Humb and McBison became Stockman's right hand men following his mutation into Black Hare, a black, hare-like mutant.
- Triceratons - A warring alien race with the appearance of a humanoid triceratops that the Turtles come into contact with when they are accidentally transported across several galaxies at the end of the first season while attempting to find the missing Master Splinter. The Turtles then become a public enemy of the Triceratons for harboring a fugitive known as the Fugitoid, who possesses a transporting technology that the Triceratons desperately want. The Triceratons wish to use Professor Honeycutt's technology to aid them in their war with Krang's army. Later in the series, the Triceratons invade earth believing that the Fugitoid is still in hiding there when in reality he has gone to live with the Utrom on their homeworld.
- Emperor Ruberx - A ruthless, corrupt, and calculating warlord of the Triceraton Republic seeking the teleportal device to use against Krang's army.
- Commander Orthor - A military commander of the Triceraton Republic who is the counterpart of General Krang when it comes to military matters. Orthor is the dedicated right hand of Emperor Ruberx, the Triceraton warlord.
- Lieutenant Gorf - Commander Orthor's second-in-command.
- Sergeant Zarx - One of Commander Orthor's underlings.
- Rico/Cobra Terror - A Brazilian street thug working for Krang and his Rock Soldiers as their spy. The Turtles interrogate him and find out where Krang and his Rock Soldiers are holding April O'Neil. During their mission to rescue April, the Turtles crash a truck full of mutagen into Krang's base. Rico is exposed to the the mutagen shortly after being bitten by a red spitting cobra. He transforms into a giant snake-like monster, Cobra Terror.