Bovine serum albumin (BSA) exists as N(pH -7.0), B(pH -9.0), and E (pH < 3.5) = isomeric forms in the solution of different pH. Acid effect on the structure of bovine serum albumin and the interaction of different structure of BSA with Ofloxacin were studied by UV-Vis and fluorescence spectroscopy. Based on the fluorescence quenching of bovine serum albumin and Förster energy transfer mechanism, the quenching constants, energy transfer efficiencies and the binding distances were determined at four different pHs. The results showed that Ofloxacin has the ability to quench bovine serum albumin fluorescence with the optimal condition of fluorescence quenching constants of 1.928 1 x 10(5) L x mo l(-1), binding distance of r = 2.55 nm and quenching efficiency of 8.63 x 10(4) L x mo x l(-1) at pH 4.9. Non-radiative energy transfer and static quenching were the cause of fluorescence quenching. The influence on the binding of Ofloxacin and bovine serum albumin under neutral, subacidity and alkalescent conditions was not obviously observed, and the electrostatic interaction was not the main force. The effect of Oflx on the conformation of BSA was also investigated using synchronous fluorescence spectrometry.