Abstract: Both of the Stremptomyces roseoflavus strain Men-myco-93-63 itself and its antibiotics produced show very strong antibiosis activities against Verticillium dahliae in the lab and field. The formation and regeneration of protoplasts of Men-myco-93-63 were developed in this study. Different effects of protoplast formation of Men-myco-93-63 were tested, including the sub-culture medium, the sub-culture time, the concentration of Lysozyme or Gly, et al. Methods for the formation and regeneration of Stremptomyces roseoflavus Men-myco-93-63 protoplasts are described: Spores shake cultured in TSB for 24h,Add ω＝2%Gly to SGGP(culture medium), continue shake cultured for 40h, Collect mycelium, Protoplasts of Stremptomyces roseoflavus Men-myco-93-63 strains were obtained by treatment with lysozyme whose concentration is 2mg/ml in a protoplast formation buffer. The protoplast of Men-myco-93-63 has been prepared successfully and Cells were regenerated on a complex medium. regenerated with 4.8×106/ml on R5.The methods were applicable to other species of Stremptomyces roseoflavus as well. It laid a foundation for protoplast transform and gene expression.