The reaction conditions for the determination of bovine serum albumin (BSA) using o-hydroxyphenylfluorone (o-HPF) as fluorescence probe reagent were studied. In the B-R medium at pH 7.90, o-HPF could react with BSA, producing a stable complex compound which resulted in fluorescent quenching of this binary system. The quantitative assay was carried out based on the relation between the degree of fluorescent quenching and the additional amount of BSA. The method has advantages of good selectivity, stability and simplicity. The linear range of o-HPF fluorescence spectrophotometric method was 1.32-18.54 microg x mL(-1). Moreover, the mechanism of the reaction BSA and o-HPF was discussed, and the quenching constant, and the ther modynamics constant of the fluorescence effects were calculated. The results showed that the nonconvalent binding forces were the binding force between o-HPF and BSA, and the quenching of BSA to o-HPF was probably a single static quenching process.