What does "I got a bee up my ass about you two" mean?

It's a quaint/inventive conflation of...

have a bug up one's ass
be very irascible and touchy
^{(Source: dictionary.reference.com)}
and
have a bee in one's bonnet
be preoccupied or obsessed with something
^{(Source: oxforddictionaries.com)}

This isn’t exactly an established idiom, but it is easily comprehensible to a native speaker.

There is an established idiom, to have a bee in your bonnet (‘bonnet’ being an old-fashioned type of headwear), which means “be preoccupied or obsessed with something” (ODO definition). Imagine having a bee buzzing around inside your hat all the time—that would make it quite hard for you to think about anything else, and you’d be(e) obsessed with it.

Then there is another established idiom, a pain in the ass (or back side, or posterior, or indeed any other term for that part of your body—can also be neck, if you’re too sensitive to refer to bums), which just means something that is really annoying. The notion presumably comes from haemorrhoids or cricks in the neck, which are constantly vexing and annoying those who suffer from it.

If you blend those two idioms, you might end up with a bee in/up your ass, which would be something extremely irritating that you can’t help but focus on exactly because it’s so irritating.

Note that Urban Dictionary even has as an existing slang term the word assbee, which would be a pithier version of bee in/up [someone’s] ass:

Jesus, this project is really turning out to be a major assbee.

– where major assbee is more or less exactly equivalent to major pain in the ass.