Genome　shuffling　was　used　to　achieve　cellulase　high　production　mutants　of　Trichoderma　reesei　T306　in　this　study.　Factors　affecting　protoplast　preparation,　regeneration　and　fusion　were　studied.　The　optimum　conditions　of　protoplast　preparation　and　regeneration　of　Trichoderma　reesei　were　as　follows:　lywallzyme　concentration　3.5%,　mycelium　age　22　h,　enzymolysis　time　4　h,　enzymolysis　temperature　30,　the　stabilizer　buffer　system　was　pH　5.0　sodium　hydrogen　phosphate-citric　acid　buffer,　regeneration　medium　was　hypertonic　screening　medium　containing　0.8　mol/L　NaCl.　Under　the　optimum　conditions,　the　protoplast　yields　and　regeneration　rate　were　up　to　1.44　×　107/mL　and　2.64%,　respectively.　Furthermore,　the　fusion　experiments　showed　that　it　was　better　to　mix　the　protoplasts　together　after　two　inactivation　methods　of　UV　irradiation　for　90　s　and　microwave　irradiation　for　200　s.　The　protoplast　fusion　rate　reached　0.5%　when　treated　at　30　for　12　min　in　the　system　composed　of　30%　PEG　and　30　mmol/L　Ca2+.　A　fusant　G2-0832　with　higher　production　of　endoglucanase　and　genetic　stability　was　obtained　after　two　rounds　of　genome　shuffling,　which　CMCase　activity　reached　86.40　U/mL,　57.08%　higher　than　original　strain.　©　2016,　Editorial　Office　of　Journal　of　CIFST.　All　right　reserved.