Abstract: Objective: The Ethanol-ammonium sulfate aqueous two-phase extraction of lutein from physalis pubescens was studied, and the antioxidant activity of lutein was further discussed. Method: The effects of ammonium sulfate mass fraction, ethanol mass fraction, and sample solution mass fraction on phase ratio, distribution coefficient, and extraction rate were examined by single factor experiment. The extraction process was optimized by Box-Behnken response surface methodology and the extraction rate as index, and the antioxidant activity of lutein was evaluated by measuring the clearance rates of DPPH free radicals and hydroxyl radicals. Result: The results showed that the optimum conditions were as follows: The mass fraction of ammonium sulfate was 12.1%, the mass fraction of ethanol was 33.6%, and the mass fraction of sample solution was 11.4%. Under these conditions, the extraction rate of lutein was 84.58%. When the concentration was in the range of 1.0~15.0 mg/mL, the IC₅₀ value of lutein from P. pubescens for DPPH free radicals was 3.26 mg/mL, and the highest clearance rate could reach to 95.32%; the IC₅₀ value of lutein from P. pubescens for hydroxyl radicals was 1.31 mg/mL, and the highest clearance rate could reach to 89.12%. It indicates that lutein from P. pubescens with strong antioxidant activity could be prepared by aqueous two-phase method. Conclusion: This study could provide a theoretical basis for the development and utilization of lutein from P. pubescens related products.