Xanthine oxidase (XO) plays a vital role in inducing hyperuricemia and increasing the level of superoxide free radicals in blood, and is proved as an important target for gout. Chrysoeriol (CHE) is a natural flavone with potent XO inhibitory activity (IC50 = 2.487 ± 0.213 μM), however, the mechanism of interaction is still unclear. Therefore, a comprehensive analysis of the interaction between CHE and XO was accomplished by enzyme kinetics, isothermal titration calorimetry (ITC), multi-spectroscopic methods, molecular simulation and ADMET. The results showed that CHE acted as a rapid reversible and competitive-type XO inhibitor and its binding to XO was driven by hydrogen bonding and hydrophobic interaction. Moreover, CHE exhibited a strong fluorescence quenching effect through a static quenching procedure and induced conformational changes of XO. Its binding pattern with XO was revealed by docking study and the binding affinity to XO was enhanced by the interactions with key amino acid residues in the active pocket of XO. Further, CHE showed good stability and pharmacokinetic behavior properties in molecule dynamic simulation and ADMET prediction. Overall, this study shed some light on the mechanism of interaction between CHE and XO, also provided some valuable information concerning the future therapeutic application of CHE as natural XO inhibitor.