No, but there was the discovery by Bartolommeo Eustachio at the time of a connection between the ear and throat which Shakespeare was probably aware of. The ancient Greeks also knew of the eustachian tube.
The use of the ear to poison Hamlet's father seems to be more metaphorical and connects to the idea of deception and betrayal. Poison is a sly, sneaky way to murder someone and by having it poured in the ear, Shakespeare seems to be alluding to the regicide of King Hamlet by Claudius as the act of an obsessively power hungry and deceptive man.
Also through there being no trace of the poisoning due to the method, Hamlet questions whether the ghost is really his father and whether he was in fact murdered. This sparks the traps that Hamlet lays throughout the play to "catch the conscience o' the king".