The article working on optimized the protoplast preparation and regeneration conditions of rare edible fungi Gloeostereum incarnatum. The conditions are defined, use mycelium cultured 7 d, and 0.6 mol/L mannitol solution was used as Osmotic stabilizer to make lywallzyme solution of 2%.At 30℃, 70 r/min Enzymatic hydrolysis of 3 hours, the largest protoplasts yield can be obtained. The regeneration rate of RM4 medium at 28℃ was the highest,regeneration rate could reach 1.83%.