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 is quenched by ADT,which is a dynamic quenching process. The binding constants Kb and the corresponding number of binding sites n are obtained by calculation. The thermodynamic parameters obtained from measured data showed that the interaction of ADT and BSA is mainly driven by the electrostatic force. The primary binding site for ADT is located at sub-domain ⅡA of BSA.The values of Hill's coefficients were nearly 1,which indicate that there is nearly no cooperative effect.The effect of ADT on the conformation of BSA is analyzed by synchronous fluorescence spectrometry. In addition, the effect of metal ions on the binding constant of ADT with BSA is also studied.