Abstract: In order to obtain high yields of the protoplasts of Streptomyces gongzhulingensis 769, this paper described an enzymatic method for that. In this study, spores of S. gongzhulingensis 769 were treated with bacteriolytic enzyme. The optimal conditions, including the concentration of lysozyme, enzymolysis time, enzymolysis temperature and the osmotic stabilizer, were studied via single-factor method. The result indicated that when the spores were treated with bacteriolytic enzyme of 3.5 mg/mL, at 36℃for 3 hours in osmotic stabilizer of P buffer, the regeneration rate of the protoplasts can reach 62.5%. And the protoplasts preparation makes important foundation for the establishing of the protoplast-mediated genetic transformation system of S. gongzhulingensis 769.