A two-stage deep eutectic solvents (DESs) treatment was shown to be an effective method for improving the utilization of certain DESs, and the specific order of pretreatment, such as malic acid/proline (MP) or choline chloride/oxalic acid (CO) during the first stage and choline chloride/urea (CU) during the second stage, resulted in better performance for enhancing the sugar yield due to the synergistic effect of the two DESs on biomass fractionation. Moreover, the presence of water during these processes could balance the loss of components by tuning the pretreatment severity, thus ensuring higher sugar yields. By eliminating the washing step after the first stage treatment, enhanced cellulose recovery and glucose yield were achieved for the CO-CU pretreatment in the presence of 5% water, and a simpler process was established with a glucose yield of 90.2% after a 3-h treatment at 100 degrees C. (C) 2017 Elsevier Ltd. All rights reserved.