Abstract: The spectroscopic characteristics of BSA and ACN interaction with the different temperatures( 25,30,35℃) in water system was investigated,using the fluorescence,synchronous fluorescence and UVvisible spectroscopy.The thermodynamic and the binding parameters( binding constants,binding distance and number of binding sites) of BSA and ACN interaction under different temperature were obtained from the Tachiya model and Stern- Volmer equation,respectively. The results showed that the intrinsicfluorescence intensity of BSA was quenched by CAN and the quenching type was static quenching. The magnitude of thermodynamic parametersΔH,ΔS for the interaction at different temperatures indicated that the electrostatic interactions played a main role in the binding of ACN to BSA. The synchronous fluorescence spectra of BSA showed that the maximum absorption wavelength of tryptophan residue shifted toward the short wavelength direction caused by the addition of ACN,indicating that the two binding sites closer to the tryptophan residues. According to Stern- Volmer equation,the binding constant decreased as the temperature increased. The number of binding siteswas not affected by temperature.Based on the Tachiya model,the binding constant and binding sites would increase with the increase of temperature.Therefore,the number of binding sitesis hardlychanges with the temperature obtained by Stern-Volmer,butthe actualnumber of binding sites increased with the increase of temperature obtained by Tachiya. So the Tachiya model could be more in line with the actual situation of the interaction between small molecule and biological macromolecule.