The chiral discrimination studies of biological system are theoretically and practically significant for the development of chiral drugs and life science. Our work has embarked upon the interaction between serum albumin (SA) (including human SA and bovine SA), R,S-1-(4-methoxyphenyl)ethylamine, and R,S-1-(3-methoxyphenyl)ethylamine. The formation of intermediate transition state, binding sites, and chiral discrimination ability can be investigated by ultraviolet-visible spectra and fluorescence spectra. Moreover, both the changes of hydrophobic microenvironment and energy transfer can be detected by synchronous fluorescence spectra and fluorescence lifetime.