The invention relates to a preparation method of a biological enzyme, particularly relates to a biological enzyme preparation method of natural brassinolide. The method includes: steps of inoculationfermentation, pressure filtration, ultrafiltration, emulsification, enzymolysis, extraction, pressure filtration, concentration, resin absorption, concentration crystallization, vacuum filtration, drying and packaging. The invention has advantages of simple process method, low energy consumption in production process, high product purity and environmental conservation.