I really don't know what the hardest boss I've ever beaten was, but some that come to mind:
Sonia - the final boss of Equinox
The Great Commander's second form on Venom - Third Path of Star Fox
Mephisto - the final boss of Alundra 2
The Reptilicus Maximus boss of Alundra
Saradiart - Final boss of Toxa's quest in Kartia
Giant Exodus Spaceship in Journey to Silius
Jaquio in Ninja Gaiden (if you don't use a special weapon)
Dracula - final boss of Castlevania 3
The crab in Blaster Master (if your gun is not powered-up)
Gruntilda - Final boss of Banjo-Kazooie
