This game's controls use a four-button scheme (two punches and two kicks, weak and strong). A particular feature is the possibility to use a super special attack. In order to achieve this, the player must fill a green bar under the life bar, by hitting their opponents. Once full, the player must press the two strong attack buttons simultaneously. There is also the option of enhancing the speed of the game, making the fights more intense but also more difficult to follow.

In addition to the main and versus modes, there is a story mode in which the Turtles must rescue April O'Neil and Splinter from Karai's clutches. The Turtles must travel across the US in their Turtle Blimp, defeating other fighters and collecting information. Only the four of them can be playable whereas the other characters (as well as a turtle clone) are the opponents. There is no Mutagen Meter in story mode. There is also a watch mode, which features computer-controlled characters.

A tournament has been organized and many fighters have entered, Shredder being one of them. The Turtles decide to participate in order to stop their nemesis as well as proving their strength in the tournament.