Abstract γ-Valerolactone (GVL)/water/acid has been developed as a mild solvent system to pretreat biomass. In this study, Eucalyptus was pretreated with 80/20 GVL/water with the addition of H2SO4 (20–100 mM) at 120 °C for 30–60 min, and the resulting cellulose-enriched residual fractions were further enzymatically hydrolyzed. Results showed that the pretreatment of Eucalyptus with GVL/water/acid system led to a significant decrease in the contents of lignin and hemicelluloses, and the enzymatic hydrolysis efficiency of the pretreated sample was significantly improved. Meanwhile, the removal of lignin and hemicelluloses from the cell walls during the pretreatment process was monitored by the confocal Raman spectra. Under the optimum conditions (50 mM H2SO4 and 60 min), the delignification rate was up to 90.7% and the cellulose content in the residue was reached to 86.1%. The high cellulose content led to a high cellulose conversion rate and the glucose yield was 6.1-fold higher than that of the raw material without pretreatment. In short, this process provided an efficient approach to remove hemicelluloses and lignin from Eucalyptus to enhance enzymatic hydrolysis for bioethanol production.