This work evaluates the effects of increasing hydrolysis time, enzyme loading and mass of substrate on the formation of glucose in the enzymatic hydrolysis of waste office paper, after chemical or physical pre-treatment. Initially, for this study, a factorial design 24 was applied. The type of pretreatment was the only significant effect on the glucose release. Maximum glucose concentration was less than 0.5 g/L, when physical pre-treatment was used. Physical pre-treatment was therefore discarded. A new factorial design with three factors and higher number of level of treatments (hydrolysis time—24, 48 and 72 h; enzyme loading—5.2, 7.8 and 10.4 FPU/g substrate; mass of waste office paper—2.25, 3.00 and 3.75 g) was applied, after chemical pre-treatment. The increase in mass of paper increased the glucose concentration. On the other hand, the increase time of hydrolysis or enzyme loading did not influence glucose release. Maximum glucose concentration (23 g/L) was about 50 times higher than that obtained when physical pretreatment was used, reducing by half the enzyme load. Optimum conditions of enzymatic hydrolysis was obtained at 24 h of hydrolysis, using lower enzyme loading (5.2 FPU/g) and higher mass of waste office paper (3.75 g; 7.5 % w/V), after chemical pre-treatment.