- 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.
- Buster/Chamelian - Buster is a street thug who joins the Shredder. As Buster was previously touched a chameleon, the mutagen transforms him into Chamelian, a lizard-like mutant with razor sharp claws, and sharp teeth.
- Harold/Mongrowl - Harold is an imprisoned Irish street thug when Shredder frees him and indoctrinates him into the Foot Clan. As Harold was previously bitten by his pet Irish Terrier, Tex, the mutagen transforms him into Mongrowl, a hunchbacked, dog-like mutant with a large hands, acute senses, and super-strength.
- Hun - Hun is the Shredder's muscle, and leader of the Purple Dragons, an urban street gang that Shredder sometimes utilizes.
- Baxter Stockman - A technical genius and the scientist responsible for creating the mousers: a series of mouse-like robots that can eat through practically anything.
- Mousers - Mousers are small robots that were created by Baxter Stockman to steal on his behalf. Stockman programs them to attack anything tagged with his radioisotope spray.
- Foot Ninjas - The foot soldiers of the Foot Clan.
- Krang - The brilliant yet ruthless Utrom scientist who has become allies with the Foot Clan. He designed a tall, powerful android that resembles a gorilla. Baxter Stockman would build Krang a robot body that use him as a head. Krang can move the robot body in synchronism as if it were his real body. When his bodies aren't in use, he uses a much smaller bipedal machine known as a "dome walker" which is equipped with several weapons.
- 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.