The fluorescence spectroscopy of bovine serum albumin(BSA)influenced by Lev-ofloxacin is studied. When the Levofloxacin is added into BSA solution gradually,the exci-tation peak has not obvious remove. However,the new phenomenon is emerged in emission spectrum. The intensity of 353.1 nm emission peak is decrease obviously,which is a typical quenching phenomenon. A new peak is emerged at 450.4 nm. The quenching progress of Levofloxacin-BSA is not dynamic quenching resulted from the molecular diffusion and colli-sion. It resulted from the static quenching progress due to the chemical component between molecules. The dissociation constant of Levofloxacin from BSA is about K_d=3.39×10^－5 mol/L.The critical distance R₀ between provider and acceptor,when energy transfer efficiency is 50%,is about R₀=1.81×10^－7cm. The energy transfer efficiency between Levofloxacin and BSA is E = 0.351.According to these calculation results,it could be known the combined position be-tween the binding site of Levofloxacin and the tryptophane of BSA is about r=2.01×10^－7 cm.