The interaction between penicillin G and bovine serum albumin was studied using fluorescence spectroscopy,synchronous fluorescence spectra,and circular dichroism spectra and incubated in pH6. 6,37℃ water bath. Static quenching was predominant between penicillin G and BSA,and non-radiation energy transfer between molecules was observed. The results showed that hydrogen bond played an important role in the binding reaction. It was found that fluorescence of tryptophan residue was quenched in the interaction of penicillin with bovine serum albumin and α-casein by Synchronous Fluorescence. The study about penicillin binding BSA by CD spectroscopy proved that the binding of penicillin and protein would change the protein conformation. It was further explained that penicillin and protein form steady compound in the cow's milk.