The thermodynamic behavior of the interaction between Quercetin and HSA is investigated by FS, UVS and MS. In fluorescence experiments, The interaction of quercetin and HSA, with C (quercetin) increased from 0 to 19.62×10-7 mol/L, the fluorescence intensity of HAS decreased from 844.3 to 461.4, the maximum emission wavelength from 340.7 nm to 338.6 nm, did not change significantly, and as the temperature increases from 298K to 303K, the quenching constant decreases from 4.66×105 L/mol to 2.69×105 L/mol the fluorescence intensity of HSA obviously decreases with the increase of quercetin concentration, but the maximum emission wavelength has no obvious displacement, and the quenching constant decreases with the raising of temperature. The results show that quercetin can strongly quench intrinsic fluorescence of HSA and quenching mechanism of quercetin with HSA is mainly a static quenching. In molecular simulation experiments, the quercetin molecule on Sudlow’s sites I and Sudlow’s sites II with the highest point are 6.41 and 7.36, in a total energy of -37.01 kJ/mol and -39.98 kJ/mol respectively, show stronger binding on site II and hydrogen bonding interactions are the major forces(bond energies are -10.41 kJ/mol and -12.34 kJ/mol respectively). And on the site I and site II, the distances from quercetin to the center position of fluorescent amino acid residues Trp214 are 1.36 and 2.40 nm, which are in agreement with the distance (3.04 nm) of calculated by the Förster’s non-radiative energy transfer theory.