Flavonoids in Valeriana officinalis L. extracted by ultrasonic-assisted aqueous two-phase extraction was utilized in this study. Effects of ultrasonic time, amount of (NH₄)₂SO₄, ultrasonic temperature and liquid-solid ratio were studied by the single factor methodologies, and then the extraction parameters were determined by Box-Behnken response surface methodology. In addition, reducing power, DPPH radical scavenging and hydroxyl radical scavenging activity were compared to investigate the antioxidant effect of flavonoids by reflux extraction. The results showed that optimized extraction parameters were ultrasonic time 35 min, amount of (NH₄)₂SO₄ 0.20 g/mL, ultrasonic temperature 51 ℃ and liquid-solid ratio 241 （mL/g）. Flavonoids yield of (6.37±0.08) % was obtained under such conditions. The extracted flavonoids showed strong reducing ability, DPPH and hydroxyl radicals scavenging activities. Moreover, the antioxidant effected by ultrasonic-assisted aqueous two-phase extraction was much higher than those of reflux extraction.