Under the simulation of the human body physiological conditions,the interaction between Adenosine Disodium Triphosphate (ADT) and bovine serum albumin (BSA) was studied with UV-visible absorption spectra and fluorescence spectra. The effects of metal ions Mn₂₊、Co₂ 、Cr₃ 、Mg₂ 、Fe₃ on the interaction of ADT with BSA were also discussed. The results showed that the fluorescence of BSA was quenched by ADT, which was a dynamic quenching process. The binding constants K_b and the corresponding number of binding sites n were obtained by calculation. The thermodynamic parameters obtained from measured data showed that the interaction of ADT and BSA was mainly driven by the electrostatic force. The primary binding site for ADT was located at sub-domain ⅡA of BSA. The values of Hill"s coefficients were nearly 1, which indicated that there was nearly no cooperative effect. The effect of ADT on the conformation of BSA was analyzed by synchronous fluorescence spectrometry. In addition, the effect of metal ions on the binding constant of ADT with BSA was also studied.