Objective This study aimed to optimize the purification process of flavonoids from Phallus impudicus and explore its anti-fatigue effect, and develop potential anti-fatigue products.Methods The purification process of flavonoids from P. impudicus was optimized using single factor and response surface methodology, and the effect of purified flavonoids from P. impudicus on exercise endurance in mice was studied.Results Compared with AB-8, HP-20, and D101, the PVPP had the highest adsorption rate for flavonoids from P. impudicus, and the optimal purification process was as follows: the concentration of crude extract of flavonoids from P. impudicus was 3.2 g/L, the concentration of ethanol elution solution was 56%, the elution flow rate was 28 mL/min, and the elution volume was 2 times the column volume. Purified flavone, i.e. (92.05±1.05)%, effectively prolonged the weight-bearing swimming time of mice, reduce the contents of serum lactic acid, blood urea nitrogen, creatine kinase, malonaldehyde and serum urea, and increased the contents of serum lactate dehydrogenase, total antioxidant capacity, glutathione peroxidase, liver glycogen and muscle glycogen in mice.Conclusion PVPP was an excellent new medium for purifying flavonoids. Purified flavonoids from P. impudicus could alleviate fatigue and enhance endurance by changing biochemical indicators, enhancing glycogen storage capacity, and antioxidant capacity. Flavonoids from P. impudicus could be used as a functional food additive.