Abstract: This work investigated the condition of macroporous resin purified of flavonoids extract from Eupatorium fortunei Turcz. and effects of anti-oxidation and resisting exercise fatigue. The optimum purification condition was confirmed by single factor experiment, meanwhile the activity of anti-oxidation and resisting exercise fatigue of different product were researched by free radical scavenging experiment and animal experiment, respectively. The results showed that the optimum purification conditions were as follows: The sample concentration was 3.0 mg/L, loading flow rate was 1.0 mL/min, the pH of sample solution was 4.0, and the volume of sample solution was 60 mL, and the 100 mL of 70% ethanol solution with 1.0 mL/min of desorption velocity. The purity of flavonoids in product increased from 24.2% to 69.4%. The anti-oxidation capacity of purified product was higher than crude extract, and the IC₅₀ values for ∙OH and O₂⁻∙were 0.47 and 0.42 mg/mL, respectively. Compared with blank control group and crude extract group, the purified product could evident prolonged exercise time of mice, decreased concentrations of lactic acid and blood urea nitrogen after exercise of mice, and possessed enhancement activities of SOD and GSH-Px, then decreased the concentration of malondialdehyde in vivo. Therefore, it had better resisting exercise fatigue effect, and the research could provide a reference for exploit of Eupatorium fortunei Turcz.