The interaction between 3, 5-dinitrobenzoic acid and bovine serum albumin (BSA) was studied by the method of fluorescence spectroscopy. By the analysis of fluorescence spectra and fluorescence intensity, it was observed that the 3, 5-dinitrobenzoic acid had a strong ability to quench the intrinsic fluorescence of BSA through static quenching process. Based on fluorescence quenching results, the apparent binding constant (K) between 3, 5-dinitrobenzoic acid and BSA was 7.08×106 and the number of binding sites (n) was 1.5 at 298 K. The distance (r) between donor (BSA) and acceptor (3, 5-dinitrobenzoic) was 4.17 nm according to the Förster’s theory of non-radiation energy transfer. The negative △H and △S in case of 3,5-dinitrobenzoic acid-BSA complex showed that hydrogen bonds and van der Waals force played a significant role in the binding of 3,5-dinitrobenzoic acid to BSA. The synchronous fluorescence spectra revealed that 3, 5-dinitrobenzoic acid induced conformational change of BSA.