Abstract: Through on cross-test experiments of protoplast formation and regeneration of Pseudomonas aeruginosa, the affects factors on protoplast formation and regeneration of P. aeruginosa were studied.The optimum conditions are as follows,0.5 hour of the treating time 2 mg/L of the concentration of lysozyme and 15 hours of incubation time. Under the optimum conditions, the ratio of formation and regeneration protoplast are 80.5% and 20.6%,respectively.