ABSTRACT. The aim of this study was to optimize the enzymatic hydrolysis of defatted rice bran (DRB) for release fermentable sugars with subsequent bioethanol production. Optimization of enzymatic hydrolysis was achieved using a sequential experimental design, performing a fractional factorial design of amyloglucosidase (AMG), at action times of two and three hours, respectively. In bioethanol production, it was evaluated the effect of adding of 15 μL g -1 of protease prior to the action of amylases. Fermentation was conducted at 29°C, with 4.0% Saccharomyces cerevisiae and initial pH of 5.0. The hydrolysis with protease yielded 3.55% ethanol, an increase of 115.15% in relation to the medium without protease addition, and the conversion had a yield of 101.5% in 24h of alcoholic fermentation. This study demonstrates the possibility to obtain bioethanol from DRB.