This work aimed at the production of cellulases from pretreated sugarcane bagasse by the filamentous fungus Trichoderma harzianum IOC 3844 and their application in the hydrolysis of this same substrate, for a future use in second-generation ethanol production. The production of cellulases was optimized, which resulted in high enzymatic activities after 42 hrs of process in an instrumented bioreactor (CMCase 27,017 U x L-1; FPase 1,225 U x L-1; and β-glucosidase 609 U x L-1). The enzymatic extract was concentrated by using a hollow fiber membrane filtration system. The concentrated extract was applied in the hydrolysis of pretreated sugarcane bagasse, after 28 hrs of enzymatic reaction, displaying a similar catalytic performance of that attained with a commercial enzymatic preparation (hydrolysis efficiency of roughly 50%). Finally, the enzymatic extract was partially characterized in terms of the molecular weights of the main activities of the enzymatic pool. Electrophoretic analysis identified eleven protein bands; six of them were related to CMCase activity and revealing molecular weights that varied from 48 to 78 kDa, and two bands were associated with β-glucosidase activity and having molecular weights of 75 and 85 kDa.