Abstract: Objective: To optimize the extraction process of Ilex latifolia Thunb. polysaccharide and analyze its inhibitory activity of xanthine oxidase. Methods: A response surface optimization ultrasound-assisted method was used to study the effects of material/liquid ratio, ultrasonic time, ultrasonic temperature and ultrasound power on the yield of Ilex latifolia Thunb. polysaccharide, and the inhibition rate of Ilex latifolia Thunb. polysaccharide on xanthine oxidase and its kinetic constant K_i were determined by in vitro experiments. Results: The optimized conditions of the response surface test were: The material/liquid ratio was 1:25 g/mL, the ultrasonic time was 60 min, the ultrasonic temperature was 40 ℃, the ultrasonic power was 260 W and the yield rate was 5.85%. The inhibition rate of xanthine oxidase by Ilex latifolia Thunb. polysaccharide was concentration-dependent with an IC₅₀ of 1.28 mg/mL, the type of inhibition of xanthine oxidase was competitive and reversible with an inhibition kinetic constant K_i of 0.62 mg/mL. Conclusion: Ilex latifolia Thunb. polysaccharide could inhibit the activity of xanthine oxidase and its could be studied as a potential food or health product for the prevention of hyperuricemia, with a view to providing theoretical data on the comprehensive utilization of Ilex latifolia Thunb..