The preparations of protoplast of Streptoverticilluim mobaraense were studied. Results showed that 4% glycine containd in culture medium were propitious to release of protolast. The optimal preparation conditions were as follows: enzymatic temperature 41 ℃, lysozyme 2.5 mg/mL, enzymatic time 4 h. Under those conditions, the product of the generation rate and regeneration rate reached 24%.