Here's some feedback I have after watching the video a few times:
- I think the player's standing animation is a bit off as he seems twitches back and fourth in his idle pose. I compared it to both the SotN and PoR "Fake Trevor" sprites and they don't seem to have this issue. Easy fix though, just adjust the hot spots to be more consistent.
- It seems like the entire boss animation takes about 19 seconds to finish, it's feels kind of long. Maybe it can be shortened a bit?
- This is a personal gripe, but I'm really not a fan of of the use of a single 16x16 block to fill in for every floor and wall in an entire room. I know you're emulating some of the older Casltevanias which do the same thing, but it looks really poor in my option. I just can't help but compare it to other SNES games like Super Metroid which had a ton of variety not only in the tiles themselves, but the placement of said tiles to give each room an organic look and flow. Seeing as how a lot of graphics you're using are from the SotN style games, I'd highly suggest taking advantage of their more diverse tilesets in cases such as this.
- Both the floor tiles and pillars behind the player layer are very.. green. It clashes with the brighter background you have behind it.
- This isn't really a problem but more of an idea. The boss seems to work pretty well but maybe you make him more of a challenge by allowing him to use sub items against the player?
Anyways, I think that's about all I can think of at the top of my head; if there's anything else I failed to mention I'll post it here later. By the way, I like the boss music!
Thanks for all the feedback and suggestions!

I fixed Trevors idle glitch lol, guess I was so used to it and focused on bigger problems I just put it off

it ended up being a hotspot problem about halfway through the animation, I just had to move it over 1 pixel and it is spot on now. As for the start up, thats an easy fix. All I have to do there is just increase the speed of the slime ball orbs and it'll be good to go. As for the backgrounds, ive been a little iffy with those myself concerning the blocks. There are several other levels that dont have those, but more of the SOTN styl you mentioned so I left it as is, kind of a nostalgia throwback lol. And thanks for the comment on the boss tune! Thats an original track I made for the game, im a huge fan of the LoS series orchestra so I tried to capture that same feel lol