Abstract: OBJECTIVE To explore the optimum extraction process of flavonoids from Isatidis Radix and evaluate its antioxidant activity. METHODS Based on the single-factor tests, the extraction conditions of total flavonoids from Isatidis Radix were optimized by response surface methodology. Meanwhile, the antioxidant activity of flavonoids was assessed by scavenging assays of 1,1-diphenyl-2-picrylhydrazyl(DPPH). RESULTS The optimum extraction conditions were as follows: ethanol concentration was 60%, liquid-solid ratio was 1∶24.1, and extraction time was 29 min. Under the optimal conditions, the extraction rate of total flavonoids was 54.4 mg·g^-1. DPPH radical scavenging capacity of flavonoids was good. CONCLUSION The target of extracting flavonoids compound efficiently from Isatidis Radix has been achieved by the optimized technology.