Abstract: To explore the optimum extraction process of total flavoniods from Cronartium orientale aeciospores and evaluate the antioxidant activity, ethanol solution was used to extract the total flavonoids and NaNO_2-Al(NO₃)₃ method was established to measure the total flavonoids rutin equivalent. Based on the single-factor tests, the extraction conditions of total flavonoids from Cronartium orientale aeciospores were optimized by response surface methodology. Meanwhile, Vitamin C was used to be a positive control on assaying and evaluating bioactivities of the total flavonoids, including total antioxidant capacity, total reducing power, capacities of scavenging 1, 1-diphenyl-2-picrylhydrazyl free radicals, scavenging hydroxyl free radical, and scavenging superoxide anion. The results showed that: 1)The optimum extraction conditions were as follows: extraction temperature 65℃, solid-liquid ratio 1:120, extraction time 38min, ethanol concentration 60%. Under the optimal conditions, the extraction rate of total flavonoids was 1.382%, which was close to the predicted value 1.416%, with 2.46% deviation. 2) In addition, total flavonoids from Cronartium orientale aeciospores possessed high total antioxidant capacity like Vc. 3) The reducing power of the total flavonoids and dose-effect relationship between reducing power and concentration were higher and clearer than Vc. 4) The total flavonoids on DPPH· scavenging capacity was lower than Vc, and the IC₅₀ on DPPH· scavenging of the flavonoids and Vc were 0.0492 and 0.0335mg/mL, respectively. 5) The flavonoids on ·OH scavenging was significant, much higher than Vc, and the IC₅₀ was 0.0129mg/mL. When the concentration was 0.0235mg/mL, the clearance could reach up to 84.94%. 6) O^2-· scavenging capacity of the total flavonoids is still unknown by xanthine oxidase method because of the interference factor in extraction solution. Reliable extraction conditions were obtained by response surface methodology and excellent antioxidant activity in vitro of the total flavonoids was verified by comparing with Vc. This could provide theoretical bases for development and utilization of rust fungi and changing concept of forest pest control.