In this study， we screened three flavonoids， Diosmetin (Dio)， Fisetin (Fis) and Genistein (Gen)， from 200 natural active small molecules using molecular docking technique combined with in vitro enzyme activity assay， and analysed the mechanism of their interactions with each other using ultraviolet spectroscopy， Raman spectroscopy and molecular dynamics. The results showed that Dio， Fis and Gen were all bound near the FAD active site of XO， and the main forces were hydrogen bonding and van der Waals force； the UV results showed that all three flavonoids had inhibitory effects on XO， and the order of the inhibitory effects was Dio>Fis>Gen； the Raman results showed that Dio， Fis and Gen increased the content of the rigid structure (α-helix and β-folding) of XO from 55.3% to 59.4%， 59% and 58.7%， respectively， and the irregular curls disappeared； DSC， particle and size potential results showed that Dio， Fis， and Gen increased the denaturation temperature Tm of XO by 3.62 ℃， 1.60 ℃， and 4.52 ℃， respectively， and the particle size decreased and the absolute value of the potentials increased； the molecular dynamics results found that the three flavonoids made the enzyme contract more by influencing the microenvironment of tryptophan residues in the vicinity of enzyme active site. The results of molecular dynamics revealed that the three flavonoids contracted the enzyme by affecting the microenvironment of the tryptophan residues near the active site of the enzyme to form a more compact and stable conformation. The experimental results showed that all three flavonoids caused the XO structure to contract to form a more stable conformation and thus inhibit the enzyme activity， and amino acid residues LEU257， ILE353 and VAL259 played a key role， which is important for the development and utilization of the novel XO inhibitor.