Abstract: The ethanol-ammonium sulfate aqueous two-phase extraction of total flavonoids from the leaves of Lonicera japonica Thunb. leaves was studied, and the antioxidant activity of the total flavonoids was further discussed. The extraction process was optimized by orthogonal test with the parameters including mass fraction of ammonium sulfate, liquid-to-material ratio, extraction temperature and extraction time as independent variables and the yield of the flavonoids as index. The antioxidant activity of the total flavonoids was evaluated by measuring the clearance rates of DPPH free radicals, hydroxyl free radicals and superoxide anion free radicals. The optimum experimental conditions were as follows: The mass fraction of ammonium sulfate was 15%, the liquid-to-material ratio was 25:1 mL/g, the extraction temperature was 50 ℃, and the extraction time was 30 min. Below this term, the yield of flavonoids was 4.98%. The clearance rate of DPPH free radicals was 97.61% when the concentration of total flavonoids from the leaves of Lonicera japonica Thunb. was 0.48 mg/mL, and those of the hydroxyl free radicals and superoxide anion free radicals were 86.53% and 75.29%, respectively, when the concentration of flavonoids was 0.72 mg/mL, showing good antioxidant activity.