Berberine is an important traditional medicinal herb, which has been effectively used in the treatment of dysentery, diarrhea, stomatitis, throat infections, and hepatitis in folk medicine. In this study, the interaction between Berberine and human serum albumin (HSA) was investigated by fluorescence spectroscopy and UV-vis absorbance spectroscopy. In the mechanism discussion, it was proved that the fluorescence quenching of HSA by berberine is a result of the formation of berberine-HSA complex. Fluorescence quenching constants were determined using the Stern-Volmer equation and Scatchard equation to provide a measure of the binding affinity between berberine and HSA. The results of thermodynamic parameters Delta G, Delta H, and Delta S at different temperatures indicate that the electrostatic interactions play a major role for berberine-HSA association. Site marker competitive experiments indicated that the binding of berberine to HSA primarily took place in subdomain IIA. Furthermore, the distance r between donor (Trp-214) and acceptor (berberine) was obtained according to fluorescence resonance energy transfer (FRET).