Duck awakes from a dream in which she is a duckling, watching a handsome prince dance ballet. Later, she walks early into class and discovers Mytho, an advanced class student, dancing alone. When she trips, he dives to catch her, twisting his ankle in the process. Duck wants to apologize to him but is prevented from doing so by Mytho's controlling roommate, Fakir. The next morning, Duck resolves to apologize to Mytho, and spies canary chicks beginning to fly. One of the hatchlings hesitates and is forced to jump when a crow tries to attack it. Mytho leaps to catch it and falls as well. To save him, Duck transforms into Princess Tutu. Mytho asks who she is. Tutu/Duck remembers that she is a duck, and runs away.

Duck and Fakir arrive at the stage where Kraehe is keeping Mytho, and Kraehe says she will give the shard of love back if Tutu confesses her feelings, knowing full well that if Tutu does she will turn into a speck of light and vanish. Tutu is prepared to sacrifice herself but Fakir stops her. He fights his way through the ravens in order to break the sword of the prince so Mytho's heart can never be shattered again. Tutu resolves to go on, and she decides to express her feelings through dance. Mytho is moved by Tutu's dance and he and the heart shard go to her, and his heart is repaired. The stage vanishes and they are lost in darkness, but a flame appears to show them the way out. The flame is Edel, who has set herself on fire to guide them back to safety. Edel makes a last request to Tutu and Mytho, that she might see them dance together. Princess Tutu and the Prince dance a duet, while Drosselmeyer muses on how they were able to overcome their tragic fate through much pain.