Efficient pretreatment is crucial for the high-value and effective conversion of lignocellulose. However, the current glycerol-based deep eutectic solvent (DES-Gly) pretreatment method requires high reaction temperatures, considerable solvent usage, and yields low conversion efficiency. In this study, we investigated the pretreatment of corn stover with 1,8-diazobicyclic [5.4.0] undeca-7-ene (DBU) synergized with DES-Gly, which significantly enhances the glucose yield from corn stover. The strong synergistic effect of DBU and DES-Gly effectively removes lignin and substantially increases glucose yield. Under optimal conditions, 95.27 % glucose conversion could be achieved, even with the addition of only 2 FPU/mL of cellulase. The physicochemical properties of the DES-Gly solution and pretreated corn stover were analyzed using FTIR, XRD, SEM, TGA, and 1 H NMR characterization techniques. In addition, DES-Gly and DBU synergy mechanisms are further explored. Our work provides a mild, efficient and low-cost DES-Gly-based pretreatment method, introducing a new way for the catalytic conversion of lignocellulose to sugar production.