Splinter was the pet rat of Hamato Yoshi in Japan who followed the teachings of his owner from his cage. When forced to America and after his master's death, Splinter found himself in the sewers of New York where he found four turtles. One morning Splinter awoke to find the turtles playing in a radiocative ooze spilling out of the canister. Soon, he noticed that not only were the turtles changing, so was he. Eventually, he grew to his fully mutated form and began to teach his adoptive sons the ways of honor and the path of the ninja. Meanwhile, the Shredder and his Foot Clan had allied himself with Krang, a ruthless, and dark-hearted Utrom from Dimension X. Much of their quest for world domination hinges on bringing the Technodrome (Krang's mobile fortress, and The Shredder's secret weapon) to the surface as it was either stuck in the Earth's core, Utrom homeworld, Dimension X, the jungles, the arctic, Arctic and Antarctic Ocean.
- 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.
- 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. He has a crush on April in this series and his brothers tease him about it.
- 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 nunchaku, which 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 - The 19-year-old college student who is a friend of the Turtles.
- 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.
- Foot Clan - A ninja organization that is led by the Shredder.
- The Shredder - One of the primary antagonists of the series and leader of the Foot Clan.
- Karai - A rebellious teen member of the Foot Clan and an expert martial artist who stirs up trouble for the Turtles.
- Krang - The most ruthless and dark-hearted Utrom who has left Utrom Homeworld and banished to the Dimension X. He designed a robot body that use him as a head. Krang can move the robot body in synchronism as if it were his real body.
- Charles Bufford/Tigerclaw - A famous martial artist and secretly a member of the Foot Clan, Charles Buford is ordered to New York with Killer to find Splinter. As Bufford was previously bitten by a caged South China tiger, the mutagen transforms him into Tigerclaw, a spike-backed, tiger-like mutant with acute senses, and super-strength.
- Killer/Reptiliar - Killer was an imprisoned Costa Rican street thug until Shredder freed him and indoctrinated him into the Foot Clan. Shredder orders this ruthless, accomplished fighter to assist Charles Bufford in finding Splinter. As Killer previously touched his pet Plumed basilisk, Dragon, the mutagen transforms him into the lizard-like mutant Reptiliar.
- Hatso/Bebop - Hatso is a nerdy African American thug who acts somewhat childish and has a fondness of toys, football, balloons, and lasagna. As Hatso previously standed closer to the caged warthog, the mutagen transforms him into the pig-like mutant Bebop.
- Greg/Rocksteady - Greg is a fat thug who has a fondness of candy bars, and cherry soda. As Greg previously sniffed by a caged rhinoceros, the mutagen transforms him into a rhino-like Rocksteady.
- Baxter Stockman - Baxter Stockman is an inventor who plotted revenge against a company called TCRI, after he was fired for spilling copier toner.
- Foot Ninjas - The foot soldiers of the Foot Clan.
- Triceratons - Triceratons are 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 a group called the Foot Clan. 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.
- Neutrinos - Neutrinos are the peaceful humanoid aliens from Dimension X.
- Princess Hanna - The beautiful princess of the Neutrinos from Dimension X whose family was attacked and killed by the Shredder and Krang. After being rescued by the Turtles, she developed romantic feelings for Leonardo.
- Professor Honeycutt/Fugitoid - A Neutrino scientist from Dimension X who was forced to take his current form after he was struck by lightning during a storm which transferred his consciousness into his worker robot.
- Utroms - Utroms are the peaceful aliens who resemble a brain.
- Mortu - Mortu was the captain of the Utrom ship that crashed on Earth. After the crash, he remained the field-leader of the marooned Utroms, and worked as their liaison to the Humans. In the modern day, this makes him CEO of the front company TCRI. His name is "Utrom" backwards.
- Dr. Sona - She was a beautiful, Utrom scientist who found and raised Leatherhead as her son.
- Leatherhead - Leatherhead is an alligator that was originally owned by a young boy until he died in a car accident and Leatherhead fall into a storm drain, where he was found by Dr. Sona and other Utroms when they discovered that their mutagen (the same that transformed the Turtles) made him sentient. Leatherhead lived with the Utroms peacefully until the Shredder's and Krang's attack forced him into hiding.
- Traximus - Traximus is Triceraton warrior and ally of the Turtles.