In no particular order...
Xemnas (Kingdom Hearts II) - To me he was hard. If you slip up, he is going to destroy you, and pretty quickly too.
Cloud Of Darkness (FFIII) - It's the fact that you have to plough through a lot to get to her, and then she is hard. If you had trouble on the preceding bosses, she is not going to let you live.
Giygas (Earthbound) - Without prior knowledge of how to beat him, you are going to be stuck for hours. Levels, especially Paula and Ness at least, should be high.
Megumi Kitaniji (The World Ends With You) - Depends what difficulty really. He is hard anyway, but on maximum difficulty, no chance!
Your rival (Pokémon Blue/Red/Yellow) - It may seem silly, but go in under prepared, and you're in for one hell of a fight. Not to mention you have to beat the Elite Four beforehand. To me, he is one of the hardest.
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