A new compound,3,8-dimethyl-4′-methoxyflavone(DMMOF) was synthesized and characterized by 1H NMR,13C NMR,IR spectra.The atomic force microscope(AFM),fluorescence spectroscopy and ultraviolet absorption spectroscopy were explored to study the interaction between DMMOF and BSA in physiological buffer solution(pH 7.4).The experiment results showed that the diameter of BSA-DMMOF was 17.705 nm while it was 2.747 nm for BSA and 2.284 nm for DMMOF.The fluorescence quenching mechanism between DMMOF and BSA was deduced to be static quenching procedure according to ultraviolet absorption spectra and Stern-Volmer equation.The binding constants and their binding sites number at different temperatures were calculated according to double logarithm regression curve.The thermodynamic parameters ΔH,ΔS and ΔG were obtained to be-116.86 kJ?mol-1,-279.55 J?mol-1?K-1and -32.14 kJ?mol-1,respectively,based on the thermodynamic equations,which indicated that the action force was mainly van der Waals force and hydrogen bonds.The binding distances(r) between donor(BSA) and acceptor (DMMOF ) was estimated to be 1.22 nm(306 K) based on the F-rster non-radioactive energy transfer theory.In addition,the effect of DMMOF on the conformation of BSA was analyzed by synchronous fluorescence spectroscopy,which indicated the addition of DMMOF will not change the conformation of BSA.Associated with ultraviolet absorption spectroscopy,the ring A of DMMOF was inferred to combine with the tryptophan-134 residues and the tyrosine residues of BSA.