Abstract: In combination with a variety of purification methods, an endo-β-1, 3-glucanase was separated from Trichoderma harzianum fermentation broth, which had high hydrolysis activity to insoluble polysaccharide curdlan. The purification process was carried out sequentially as follows, 50% ammonium sulfate precipitation from cell culture supernate, HiPrep 26/10 desalting gel chromatography, removing impurities protein by QFF anion exchange column, finally was purified by Superdex 75 gel chromatography. The enzyme activity yield was 6.63%. The purification scheme resulted in an increase in the specific activity (from 6.86 to 133.01U/mg) and a 19.39-fold purification of endo-β-1, 3-glucanase relative to the crude broth. Identified by MALDITOF-TOF, the result showed that the purified enzyme had high homology with the endoglucanase from Trichoderma virens.