Is ty important in this sentence can't you just say 'nejsi holka'?
Is the reason we add tu the same as adding ty in front of jsi?
The correct answer is with "nesji" but it refuses "nejste". I think "nejste" is also correct if you don't know the person very well.
"Ty nejsi holka" and "Vy nejste holka" are both correct.
'jseš/seš' is not accepted for 'you', why is that?
Because it is not Standard Czech but Common Czech.
Is it grammaticaly correct to write "holka nejsi ty" to make it sound more like a question ?
No, that is a wrong question.
i had the option of 'nejsi ono děvče'. Would this not be acceptable as well as a nuetral?
No. The ONO could only mean THAT. And most often it neans just IT.
So with the meaning we only show the people it is juat nonsense. With the meaning that actually somehow works it means "Aren't you that girl?".