Abstract: The main factors affecting the ultrasonic-assisted extraction efficiency of solvent types, times, ratios of solvent to material, duration and temperature on the yield of ergosterol in Flammulina velutipes were researched respectively by singlefactor experiments, and the extraction process were optimized by orthogonal tests.The results showed that the factors affecting the extraction rate of ergosterol were in the order of extraction duration, extraction ratios of solvent to material, extraction times and extraction temperature.The optimum extraction conditions were as follows:the extraction time 20 min and number of extraction 2 times, the ratio of material to ethanol was 1∶50 (g/m L) , extraction temperature 50℃.Under these conditions, the average extraction yield of ergosterol was 2.28 mg·g^-1.The linear range of ergosterol was 10.00¹00.00μg·m L^-1 (R²=0.9998) , the average recoveries (n=9) was 100.31%with RSD 2.69%, and the analysis cycle was within 11 minutes.This method showed significantly higher extraction yield and reproducibility compared with conventional reflux.