The series was 4Kids Entertainment's first fully in-house animated production, announced in May 2002 for the FoxBox programming block.

Producer Lloyd Goldfine, a longtime fan of the original Mirage Studios comics, pushed to make the show closer in tone to the comics' darker take on turtles raised as ninja assassins rather than the purely comedic 1987 cartoon, while still keeping it family-friendly.

Once 4Kids showed interest in the franchise, Goldfine suggested going directly to Mirage Studios for guidance, leading the team to visit the company's headquarters in Northampton, Massachusetts, where co-creator Peter Laird and CEO Gary Richardson approved the pitch.

Mirage Studios stayed closely involved throughout production, with Peter Laird reading every outline and script draft and approving most of the character designs.

The 'Ninja Tribunal' season was originally planned as the fifth and final season, but the airing schedule was rearranged to boost interest, making 'Fast Forward' air as season five instead while 'Ninja Tribunal' was delayed and later promoted as 'Teenage Mutant Ninja Turtles: The Lost Episodes' in 2008.

For its final season in 2008, the show moved from Fox to The CW, and the entire cast received another visual redesign based on the character designs from the 2007 TMNT film.

The finale film Turtles Forever served as a crossover between the 2003 series and the 1987–1996 animated series, using a multiverse plot device to bring both versions of the Turtles together.

The Justice Force superhero team featured characters that were direct spoofs of famous DC and Marvel heroes, including parodies of Superman, Batman, Flash, Green Lantern, Plastic Man, and Aquaman/Namor.