The work aims to optimize the ultrasonic extraction process of flavonoids from Podocarpus macrophyllus seeds by response surface methodology and investigate the antioxidant activity of flavonoids. Based on the single factor test, with the material liquid ratio, ultrasonic time and ultrasonic temperature as independent variables and the yield of flavonoids and DPPH radical scavenging rate as dependent variables, the best extraction process conditions were determined by three-factor and three-level response surface analysis. The optimal process parameters were ultrasonic frequency of 40 kHz, ultrasonic power of 200 W, material liquid ratio (g/mL) of 1:50, ultrasonic temperature of 70 ℃ and ultrasonic time of 2.7 h. The theoretically predicted yield of flavonoids was 27.54 mg/g (n=3, relative standard deviation of 1.63%). When 0.50 mL of flavonoids extract diluted 10 times was added, the DPPH radical scavenging rate was 87.2% and the reduction rate of Ce (Ⅳ) was 84.2%. When 0.90 mL of flavonoids extract diluted 10 times was added, the scavenging rate of OH was 88.1%. The ultrasonic assisted extraction process optimized by response surface methodology is stable and reliable, and can be used to extract flavonoids from Podocarpus macrophyllus seeds. The flavonoids of Podocarpus macrophyllus seeds can be used as excellent natural antioxidant resources.