The invention provides a preparation method of erythromycin by using a bioenzyme, which comprises the following steps: (1) inoculating a red streptomycete strain into a primary seed culture medium, and culturing to obtain a primary seed solution; (2) inoculating the primary seed solution into a secondary seed culture medium, and culturing to obtain a secondary seed solution; and (3) inoculating the secondary seed solution into a fermentation culture medium, and culturing for some time while supplementing bean oil, lipase, corn steep liquor, proteinase and other substances, thereby obtaining the high-erythromycin-content fermentation liquid. By controlling and optimizing the fermentation culture medium and culture process and especially introducing the bioenzyme, the preparation method enhances the raw material utilization ratio and erythromycin fermentation level, simplifies the technique and lowers the production cost.