Shenmai injection is compound traditional medicine and widely employed in adjuvant therapy of cancer patients for improving the patient′s life. The interaction of Shenmai injection with human serum albumin(HSA) in physiological buffer(PH 7.4) was investigated by fluorescence spectroscopy and UV-Vis absorption spectroscopy. These results have significant importance for understanding the pharmacological action pesticide effect of Shenmai injection. Shenmai injection can effectively quench the intrinsic fluorescence of HSA and the results shown that the quenching mechanism was a dynamic process, which was further proved by the UV-Vis absorption spectroscopy. The binding constant KA at different temperatures(296, 303, 310 K) were obtained from Modified Stern-Volmer analysis of the fluorescence quenching data. The thermodynamic parameters were calculated by Van′t Hoff equation(ΔG<0, ΔH>0, ΔS>0). It indicated that the hydrophobic interactions play an important role in the interaction of Shenmai injection and HSA. In addition, the binding process was spontaneous. Furthermore, the synchronous fluorescence spectra showed that the maximum fluorescence peak of tyrosine residues changed which meant the binding of Shenmai injection to HSA mainly acting on tyrosine residues and the interaction can induce the microenvironment and conformation changes of HSA.