To separate and purify the total flavonoids in Gynura and study its antioxidant activities in vitro and in vivo. The total flavonoids were preparated by 80% alcoholic. After removing impurities, the crude extracts were purified with type AB-8 macroporous adsorption resin. The content of total flavonoids was determined by spectrophotometry. The antioxidant activities in vitro of the total flavonoids were analyzed by using different assays compared with Vc, such as DPPH radical (DPPH•) scavenging assay, hydroxyl radical (•OH) scavenging assay, superoxide radical (O2-•) scavenging assay. The antioxidant activities were tested with the purified flavonoids in vivo. The content of total crude flavonoid from Gynura was 13.928 mg/g, and the purification ratio was 32.79%. The radical scavenging effect was enhanced with increasing flavonoid concentration. The scavenging effect of the purified flavonoids was greater than the crude extracts. The level of superoxide dismutase (SOD) activity of mice brain and liver was higher significantly in low doses groups than in normal control groups (p < 0.05), so was the level of catalase (CAT) activity of liver.The brain malondialdehyde (MDA) content was obviously lower than normal control groups(p < 0.05).