Hydrolysis of cellulose is a fundamental step related to the amount of glucose obtained for ethanol production. The aim of this work has been to improve the conditions of enzymatic hydrolysis of the study performed by Mesa, 2010. The same factors used in the study were taken into account to be improved, which are: temperature, solid percentage, Tween 80 surfactant, agitation speed, amount of cellulase and time. The Plackett-Bürman method was used for 8 experiments, to discard variables that do not influence the enzymatic hydrolysis process, in order to subsequently adjust the model and use the Box-Hunter factorial optimization design. The glucose yield was improved, obtaining results of 25.90%, unlike the first study in which 24.33% were obtained; this data for every 100 grams of bagasse.