Due to the intact structure of lignocellulosic biomass, pretreatment was a prerequisite to improve the enzymatic hydrolysis by disrupting the recalcitrant lignocellulose and increasing the accessibility of cellulose to enzyme. In this study, an alkaline hydrogen peroxide (AHP) pretreatment of sugarcane bagasse with various loadings of H2O2 (1.25–6.25 wt%) at temperatures of 60–160 °C was proposed to degrade hemicellulose/lignin and improve the enzymatic digestibility. It was found that increasing H2O2 loadings during pretreatment lead to the enhancement of substrate digestibility, whereas the alkali (only NaOH)-pretreated solid generated higher glucose yield than that pretreated under AHP pretreatment with lower loading of H2O2. This enhancement of enzymatic digestibility was due to the degradation of hemicellulose and lignin. Furthermore, Tween 80 was added to promote enzymatic digestibility, however, the increased yields were different with various substrates and hydrolysis time. The highest glucose yield of 77.6% was obtained after pretreatment at 160 °C for 60 min with 6.25% H2O2 and the addition of Tween 80, representing 89.1% of glucose in pretreated substrate. This study demonstrated that the AHP pretreatment could greatly enhance the enzymatic saccharification. The addition of Tween 80 played remarkable performances in promoting the glucose yield during enzymatic hydrolysis by stabilizing and protecting the enzyme activity. This study provided an economical feasible and gradual process for the generation of glucose, which will be subsequently converted to bioethanol and bio-chemicals.