Formation and regeneration conditions of protoplasts for two Aspergillus strains were optimized,the effect of material,cell age,osmotic stabilizer,enzyme proportion,enzyme acting time and acting temperature on the formation and regeneration of protoplasts from Aspergillus oryzae 3.951 and Asperigullus oryzae RIB40 were studied.The optimized conditions of A.oryzae 3.951 were: mycelial as the preparation material and its age was 14 h,0.8 mol/L NaCl as the osmotic stabilizer,enzyme solution has 1.0 %(w/v) cellulase,1.0 %(w/v) lysing enzyme and 0.1 %(w/v) snailase,enzyme acting time 3 h and enzyme acting temperature 35 ℃.The optimized conditions of A.oryzae RIB40 were the same as A.oryzae 3.951 but a better age of its mycelial was 12 h.Under optimized conditions,A.oryzae 3.951 released 6.43×10~7 protoplast/g with the regeneration rate of 23.5 %,and A.oryzae RIB40 released 4.24×10~7 protoplast/g with a regeneration rate of 22.41 %.