Abstract: In this study, ultrasound-assisted aqueous two-phase extraction (UA-ATPE) was adopted to extract flavonoids from Rosa roxburghii Tratt and investigated its inhibitory effect of this flavonoid on xanthine oxidase activity in vitro. In this experiment, flavonoids yield of Rosa roxburghii Tratt was taken as the index to verify the single factor and response surface optimization of flavonoids extraction process. The optimal extraction process was obtained as follows: Under the condition of 32% C₂H₅OH-20% (NH₄)₂SO₄ two-phase aqueous extraction system, the yield of flavonoids was 140.57±1.78 mg/g when the solid-liquid ratio was 1:59 (g/g), the ultrasonic time was 30 min, the temperature was 50 ℃, and the power was 320 W. Under these conditions, the IC₅₀ value of xanthine oxidase was 12.72 mg/mL in vitro, indicating a good inhibitory activity. The experiment showed that the optimized technology of UA-ATPE extraction of flavonoids from Rosa roxburghii Tratt effectively improved the yield of flavonoids from Rosa roxburghii Tratt. And the flavonoids extracted from Rosa roxburghii Tratt had a good effect of inhibiting the activity of xanthine oxidase and could be used as a natural xanthine oxidase inhibitor.