The final scene, pure and simple. Seeing Bruce stand on the building edge looking out into the night and knowing that he is and always will be Batman. Then the music swells up and the bat signal lights up the sky. Bob Kane and Bill Finger's vision perfectly displayed.
By the same note, the birth of Batman. The awesome music mixed with a simultaneous usage of Tear Jerker makes it probably the most incredible showing of his first donning of the cowl ever.
The fact that Siskel and Ebert gave the movie "2 thumbs up" and -almost- gave them good reason to turn their backs on the Animation Age Ghetto.
Even more awesome this is the ONLY Batman film of the 80s/90s to get "2 thumbs up", yes that's right, this animated Batman film is the only Batman film that Roger Ebert liked until The Dark Knight Trilogy'' note Gene Siskel liked the first three, and hated Batman and Robin, and he said MOTP was just below the first in terms of quality
Mark Hamill says that this movie was where he truly developed his repertoire of Joker laughs. A collection he used for nearly 20 years before stepping down from the role.
Joker murdering his former boss, then taking on Batman and holding his own when the Phantasm arrives to kill him.
Bruce running up the front of a motorcycle and punching the thug off in one smooth motion.