The invention provides a preparation method of ergosterol, which comprises the following steps: carrying out ultrasonic treatment, saponification and column chromatography purification on a strain, and recrystallizing twice to obtain the ergosterol product. The extraction rate of ergosterol can reach 1.63%, the purity is highe r than 99%, and the content is higher than 95%. According to the method, the advantages of ultrasonic wall breaking and saponification extraction are combined, the two operations are innovatively carried out step by step, and the extraction rate is greatly increased. Inaddition, tests find that in the purification process, recrystallization is carried out twice after column chromatography is finished, and the product purity is the highest and is remarkably superiorto that of one or three times of recrystallization. According to the whole ergosterol preparation method, most of the used solvents can be recycled, the production conditions are mild, batch production is easy to realize in factories, the cost is low, the efficiency is high, and the product is high in quality.