In order to quickly determine the content of ergosterol in waste mycelium, this paper examined the ultraviolet spectrophotometry. The extraction solvent was dichloromethane. The calibration curve was linear over the concentration range from 0.005 to 0.025 mg/mL, with a correlation coefficient of 0.999 79. The average recovery was 98.87% ~103.65%, and the relative standard deviation (RSD) range was 2.03% ~3.07%. The sample was stable within 10 hours. This method has the characteristics of simple equipment operation and rapid detection, which are suitable for immediate detection. The method has good safety, precision and stability, and can be used for the rapid determination of ergosterol in waste mycelium.