The interaction of human serum albumin and folic acid was studied using fluorescence spectroscopy, UV absorption and synchronous fluorescence spectroscopy in the pH 7.4 Tris-HCl buffer system at different temperatures. The research shows that these interactions result in the endogenous fluorescence quenching of HSA, which belongs to a static quenching mechanism. The quenching rate constants, the binding constants and the binding sites of the static quenching were calculated. The distance between the body (HSA) and receptor (folic acid) and the efficiency of energy transfer were obtained to be 1.77 nm and 0. 052 65 respectively, based on the theory of Forster nonradiative energy transfer. And according to the thermodynamic parameters calculated the binding of HSA and folic acid is mainly attributed to the hydrophobic interaction, partly static force. Further more the synchronous fluorescence spectrum was utilized to investigate the conformational transformation; The decline result of the hydrophobic nature around Trp demonstrates that the folic acid is in the hydrophobic cavity of HSA.