The reactions were studied for the determination of bovine serum albumin (BSA) using o-hydroxyphenylfluorone (o-HPF) as fluorescence probe reagent with fluorescence spectrophotometry. In the B-R medium, o-HPF reacts with BSA to form the stable complex compound. The reaction system of o-HPF and BSA was binary system, and the method for the determination of BSA was of good selectivity and stability and simplicity. The linear range of o-HPF fluorescence spectrophotometric method was 1.32-18.54 microg x mL(-1) The quenching constant and thermodynamics constant of the fluorescence effects between BSA and o-HPF were calculated. The results showed that the non-covalent binding forces were the binding force between o-HPF and BSA.