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This experiment was designed to opti‐ mize the extraction process of total flavonoids from Drynariae Rhizoma (DR) in ultrasoundassisted PEG400- (NH4)2SO4 aqueous two-phase system and to evaluate its antioxidant activity. Based on the single-factor approach, the optimal extraction process of total flavonoids from DR was optimized by response surface methodology with (NH4)2SO4 mass fraction, PEG400 mass fraction, ultrasonic power and ultrasonic time as independent vari‐ ables and the yield of total flavonoids from DR as dependent variables. The scavenging ability of DPPH· and ·OH and the iron reducing power of total flavonoids from DR were determined by using Vitamin C as a positive control. The results showed that the optimal extraction conditions for total flavonoids from DR were PEG400 mass fraction 19%, (NH4)2SO4 mass fraction 19%, ultrasonic power 180 W, and ultra‐ sonic time 40 min. The yield of total flavonoids from DR under this process was 182.98 mg/g, which was similar to the theoretical prediction. The analysis of antioxidant activity showed that the scavenging abil‐ ity of DPPH·, ·OH and iron reducing power of total flavonoids from DR increased and stabilized with the increase of concentration in a certain concentration range. The PEG400- (NH4)2SO4 aqueous twophase system was used for the extraction of total flavonoids from DR, and the optimal process conditions were reliable. [ABSTRACT FROM AUTHOR]