The aim of this research was to evaluate the anti-exercise-fatigue and promotion of sexual interest of total flavonoids from drone pupae of wasps. DPTF was prepared by ethanol extracting and its extraction conditions were optimized by response surface methodology. Then, anti-exercise-fatigue and promotion of sexual interest of DPTF were evaluated. The optimums extraction conditions by RSM were ethanol concentration 65%, extraction time 3 h and solid-to-liquid 20:1(mL/g). No mortality and general symptoms of toxicity were observed in the DPTF treated mice(1 g/kg,3 g/kg,5 g/kg body weight) the body weight and food consumption were not significantly changed compared with the normal control group. The relative weights of main organ did not markedly change. DPTF can significantly extend the duration of the swimming time to exhaustion and the times of capture the female in mice, decrease BUN, LAC and Cr levels, increase LG, GG and T activities in the DPTF treated mice. The dose of 5 g/kg body weight is the optimal dose for anti-exercise-fatigue activity and promotion of sexual interest in male mice. In conclusion, DPTF is promising traditional natural-based therapeutic remedy for relieving exercise-fatigue with high safety.