Abstract: Bacillus subtilis strain R31 could be used to control banana Fusarium Wilt in fields， and could not be genetic operated by conventional preparation of competent cells， so researching its genetic transformation method could be benefit for the understanding of its molecular bio-control mechanism. Based on the growth curve and growing status of strain R31 in LB media， the culture time suit for protoplast preparation and regeneration was chosen. The growth curve study showed that in LB media， the growth of R31 was in the logarithmic phase from 3 h to 6 h after inoculated， and it was at the end of logarithmic phase after 4.5 h. After 7 h， the growth of R31 was in the stationary phase. Observations by microscopy discovered that R31 growing as free-floating planktonic mode at the early stage of the logarithmic growth， but bacteria began to connect with each other after 5.5 h and gradually formed a net structure， then formed a three- dimensional structure at 8 h. Free-floating planktonic cells were suit for the protoplast preparation and regeneration， but gave consideration to the concentration of cells for protoplast preparation， the optimized culture time was 4-4.5 h， under this condition， and the best lysozyme concentration was 3.5 mg/mL.