Abstract: The main factors affecting the ultrasonic-microwave synergistic extraction efficiency of extraction time,microwave power,and ultrasonic power on the yield of ergosterol and vitamin D₂(VD₂)in shiitake mushroom powder were investigated by single factor and orthogonal experiments. The results showed that the factors affecting the extraction efficiency of ergosterol and VD₂ were as follows:Extraction time>microwave power>ultrasonic power. The optimal extraction conditions were absolute ethanol as extraction solvent,extraction time was 20 min,ratio of material to liquid was 1:20 (g/mL),the extraction temperature was room temperature,the ultrasonic power was 400 W,and the microwave power was 200 W. Under the extraction conditions,the ergosterol content was as high as(3.59±0.69) mg/g,and the VD₂ content was as high as(123.52±0.69) μg/g. The average recovery of ergosterol(n=6)was 114.57%,RSD(n=6)was 3.83%,VD₂ average recovery(n=6)was 102.26%,RSD(n=6)was 4.95%. The method showed higher extraction efficiency,shorter extraction and analysis time,simpler operation,and better accuracy and reproducibility compared with reported methods.