The effects of key parameters on the preparation and regeneration of protoplast from the β-carotene-producing fungus Blakeslea trispora were discussed in this paper, including the combination of various enzymes, mycelial age, digesting time and temperature, pH value, osmotic stabilizers, pretreatment, culture medium and culture method. Under the condition of mixed enzymes in osmotic stabilizer (0.6 M NaCl) combined with 2% lysozyme, 3% cellulase and 3% snailase, the highest protoplast yield, as high as 7.48×10^(6) protoplasts/mL, was obtained when mycelial age was 60 h at pH 5.0-6.0 with digesting for 14-16 h at 28 ℃. After purification of the obtained protoplasts, they were regenerated in PDA regenerative medium using bilayer plate culture method. To validate the usability of the protoplasts, a novel plasmid with green fluorescent protein (GFP) was used in transformation for easy visual observation. The results showed that the protoplasts prepared by the optimized method were active and applicable in further gene manipulation experiments.