OBJECTIVE: To optimize the extraction technology of polyphenols from Juglans regia branch and evaluate its antioxidant activity in vitro. METHODS: Using extraction amount of polyphenols from J. regia branch as response value, solvent-solid ratio, extraction temperature and ethanol volume fraction as response factors, based on single factor test, response surface method was used to optimize the extraction technology of polyphenols from J. regia branch. Using vitamin C as positive control, scavenging on hydroxyl radicals and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radicals and total reducing activities of polyphenols from J. regia branch were investigated. And its antioxidant activity in vitro was evaluated. RESULTS: Optimized extraction conditions for polyphenols from J. regia branch was as follow as solid-liquid ratio of 1 ∶ 25 (g/mL), extraction temperature of 50 ℃, ethanol volume fraction of 70%. The extraction amount of polyphenols from J. regia branch was 9.30 mg/g (RSD=0.57%, n=3) in the verification test. Clearance rate on hydroxyl radicals and DPPH radicals and total reducing activity of polyphenols from J. regia branch were respectively 50.24%, 95.42%, 1.118 when it was under the mass concentration of 12.0, 3.0, 3.0 μg/mL; and the related data of vitamin C was 93.71%, 46.17%, 0.628 under the same mass concentration (P<0.05). CONCLUSIONS: Extraction technology of polyphenols from J. regia branch optimized by response surface method is stable and feasible; polyphenols from J. regia branch shows certain antioxidant activity in vitro.