In this study, deep eutectic solvent (DES) consisted of choline hydroxide and glycerol (ChOH/G) was developed as pretreatment reagent for sugarcane bagasse (SCB). The influences of pretreatment parameters on enzymatic hydrolysis were analyzed, such as the quality ratio of ChOH to glycerol, pretreatment time and temperature. For the highest total reducing sugar yield (TRSY) and total reducing sugar concentration (TRSC), the pretreatment parameters were optimized by response surface method. After optimization, the TRSC was 17.36 g/L (Optimized-P1: quality ratio of 1:3, 170, 4 h). The TFSY was 0.51 g/g (Optimized-P2: quality ratio of 1:2, 150, 5 h).