We used sodium chlorite followed by sodium hydroxide as a two-stage pretreatment of cassava stem for removal of lignin and hemicellulose to obtain a substrate with high cellulose content prior to hydrolysis. Response surface methodology was applied to determine the optimum hydrolysis conditions of two-stage pretreated cassava stem. After pretreatment, the cellulose content of cassava stem increased from 42.10% to 86.45%, concomitant with decreases in lignin (87.59%) and hemicellulose (78.18%) content. Acid hydrolysis of two-stage pretreated cassava stem under optimum conditions allowed obtaining a hydrolyzate rich in reducing sugar, with a yields up to 67.37%. Conversely, inhibitors were detected at very low concentrations. The fermentation of the hydrolyzate resulted in an ethanol yield of 22.58 g/100 g substrate corresponding to a theoretical ethanol yield of 84.41%. The results demonstrate that two-stage pretreatment is effective for improving cellulose hydrolyzability, resulting in high fermentable sugar and low fermentation inhibitor concentrations.