Abstract: Sporecarp of Trametes cinnabarina was collected in July 2016 in Liangshui National Nature Reserve of Heilongjiang province, and wascut out on PDA agarslantculture-medium under 30℃ for 7 days. Mycelium was inoculated on liquid nutrient medium for protoplast preparation andregeneration test. Single factor and orthogonal test was carried out on enzymolysis temperatures and enzymolysis time, mass fractions of hyphae andenzyme ratios. The result demonstrated that the best condition for protoplast preparation was 0.6 mol·L-1 mannitol solution as buffer, 1.5% oflywallzyme, 1.0% of cellulase and 1.0% of glusulase as enzume ratio, 20% of mass fraction of mycelia, 3.5 hours foren zymolysis under 30℃. Theyield of protoplast topped to 1.0×107 mL-1. The highest regeneration rate was 20.6% with 0.6 mol·L-1 sucrose solution as the regeneration stabilizer.