(question stolen from InsideGaming twitter)
There are plenty of boss fights that are memorable to me, from Dark Dragon in Shining Force 1 to Mr. X from Streets of Rage 2 to Frank Fontaine in Bioshock. All of them had something different about them that I enjoyed.
However, I'm left to choose only one, and that would have to be GLaDOS from Portal. I started the game loving the cheerful little voice that came on in the room of each test chamber, helping to guide me through each puzzle and lead me closer to the prize of cake. However, each success led to increasingly dissuading remarks coming from my supposed helper. GLaDOS began to care less and less about my success, becoming bitingly apathetic towards my well-being as she led me into a firey pit. Someone who I once trusted has now become the thing I must destroy to get out of this terrible place.
Similiar occurrences happen in Halo 3 (with Cortana-Gravemind) and Bioshock (Frank Fontaine/Atlas), yet they never seemed to drive home as hard as GLaDOS did. There's no option to destroy Cortana herself (although she's right on the edge of Rampancy anyway), and Atlas/Fontaine gives himself away instead of denying it all or calling it Ryan's ploy to cloud the eyes of the player.
Or maybe it's simply because of GLaDOS' childlike voice that gets me.