Neurospora crassa NC-1-6 is a filamentous fungi strain with high cellulose activity and β-carotene production.To establish protoplast-mediated genetic transformation system of Neurospora crassa,the major factors influencing protoplast isolation and regeneration of Neurospora crassa were investigated,such as the mycelium incubation time,various enzymes and reaction time.The results showed that the mycelia incubated in liquid PDA medium at 30℃for 10 h were more suitable for protoplast release,the macerozyme solution of 0.5%(W/V)Snailase,0.5%(W/V)Lysing enzyme and 1%(W/V)Cellulase was more effective for protoplast preparation,and the suitable incubation time with macerozyme solution for the protoplasts regeneration rate was 10 h.When 0.25g wet mycelium were incubated with 5ml macerozyme solution under these condition,the number of protoplasts release and regeneration was 4.53×106 and 1.40×106,respectively.