The interaction between palmatine hydrochloride with human serum albumin (HSA) was investigated by fluorescence quenching technique and UV/vis absorption spectroscopy. The results of fluorescence titration revealed that palmatine hydrochloride could strongly quench the intrinsic fluorescence of HSA by static quenching and nonradiative energy transferring. The electrostatic interaction plays a major role in stabilizing the complex. The binding site number n and apparent binding constant KA, corresponding thermodynamic parameters DeltaG, DeltaH and DeltaS at different temperatures were calculated. The distance r between donor (HSA) and acceptor (palmatine hydrochloride) was obtained according to fluorescence resonance energy transfer. The effect of palmatine hydrochloride on the conformation of HSA was analyzed using synchronous fluorescence spectroscopy.