In order to explore the combined inhibitory effect of diosmetin (Dio)， fisetin (Fis)， genistein (Gen) and allopurinol (Allo) on xanthine oxidase (XO)， the optimal combination ratio was explored by in vitro enzyme activity determination combined with combined action index， and the interaction mechanism between them and XO was analyzed by fluorescence spectroscopy， infrared and molecular docking techniques. The results showed that the optimal molar ratio of Dio-Allo， Fis-Allo and Gen-Allo was 2∶1， and the combined inhibitors showed synergistic effect. The IC50 values were (1.45±0.13)， (5.71±0.06) μmol/L and (6.29±0.09) μmol/L， respectively. The fluorescence results showed that the combined inhibitors affected the microenvironment of tyrosine and tryptophan residues， and the binding constants Ka were 7.40×105， 6.03×105 L/mol and 4.20×105 L/mol， respectively. The infrared results showed that the combined inhibitors increased the percentage of α-helix， β-turn and random coil of XO， and decreased the content of β-sheet by 55.8%， 55.6% and 62.0%， respectively. Molecular docking results showed that Dio， Fis and Gen bind to Allo at different active sites. Studies have shown that Dio， Fis and Gen could be used in combination with Allo to achieve better inhibition of XO activity. This was of great significance for the development of new XO combined inhibitors and the reduction of the dosage of drugs with adverse reactions.