Abstract: Macroporous resin was used to purified flavonoid extract from Zingiber mioga（Thunb.） Rosc, the difference in static adsorption and elution properties between resins was compared, and the optimum purification conditions were confirmed by dynamic adsorption and elution tests after determining adsorption mechanism of the best resin type. Meanwhile, the anti-fatigue activity of the purified flavonoid extracts was investigated by animal experiments. The results showed that AB-8 macroporous resin had the best performance for flavonoid extract purification. The saturated adsorption amount gradually increased with sample concentration increasing, while decreased with the temperature increasing. The adsorption isotherm accorded with the Langmuir model. The optimum purification conditions were as follows: sample concentration 6 mg/mL, sample volume 60 mL, sample pH 6.0, loading flow rate 3 mL/min, eluent 60% ethanol, eluent volume 150 mL, and elution flow rate 2 mL/min. Under these conditions, the total flavonoids purity increased from 11.25% to 47.52%. The results of animal experiment showed that medium and high dose of purified extracts could significantly prolong exhausting swimming time of mice, decrease lactic acid and urea nitrogen contents in serum after exercise, and enhance activity of lactic dehydrogenase.Therefore, flavonoid of Zingiber mioga（Thunb.） Rosc could relieve exercise fatigue.