Regenerated cellulose can be prepared by treatment with an ionic liquid (IL) and an anti-solvent such as water, which significantly enhances the enzymatic hydrolysis in comparison to crystalline cellulose. The IL-aqueous two-phase system (IL-ATPS) is consisted of IL-condensed top phase and salt-condensed bottom phase, which could be suitable to produce regenerated cellulose with smaller amount of IL. Using IL-ATPS with different pH, the enzymatic saccharification efficiency of crystalline cellulose was determined. The use of 1-allyl-3-methylimidazolium chloride resulted in relatively higher yield of glucose production as compared to 1-butyl-3-methylimidazolium chloride. The IL-ATPS showing optimal pH for cellulase was prepared with mixed salt (NaH2PO4/Na2HPO4 = 5/1 (wt/wt)), which provide a regenerated cellulose with the pH range of 4.8-4.9 in enzymatic reaction mixture. Using such regenerated cellulose as feed of saccharification, the final yield of glucose was about 70%. (C) 2020, The Society for Biotechnology, Japan. All rights reserved.