Fermentable sugars are a group of key intermediate compounds and pretreatment is the most crucial step for the saccharification of lignocellulose. In this manuscript, microwave-assisted glycerol pretreatment was performed before fast pyrolysis and enzymatic hydrolysis to obtain fermentable sugars from corncobs. Owing to demineralization, delignification, and concentration of crystalline cellulose via pretreatment, the yield of levoglucosan (44.5%) was significantly increased as compared to that of un-pretreated corncobs (6.9%) in fast pyrolysis. Furthermore, the microwave-assisted glycerol pretreatment also facilitated glucose production in enzymatic hydrolysis. The yield of glucose increased from 19.9 to 52.1%, which could be ascribed to the elimination of lignin by pretreatment. These results demonstrated that microwave-assisted glycerol pretreatment was an economically attractive route to boost the final production of fermentable sugars from lignocellulose via following fast pyrolysis and enzymatic hydrolysis.