Choline acetate, a totally bio-derived ionic liquid (IL), was used for IL-assisted pretreatment and subsequent fractionation of bagasse into carbohydrate and lignin. The fractionated carbohydrate-rich material (CRM) and lignin-rich material (LRM) were 79.6% and 5.2% of the original bagasse, respectively. CRM contained nearly all carbohydrate and approximately 50% of lignin in the original bagasse. In addition, approximately 20% of lignin in the original bagasse was fractionated as LRM. The cellulose saccharification percentage was over 95% for CRM, although the value was only 20% for the original bagasse. The average molecular weight of LRM was 4.2×104, which was approximately ten-times lower than that of the hydrolysis residue of CRM. After five cycles of the processes, the recovery of choline acetate was 80% of the fresh one. Throughout the five cycles, choline acetate maintained its chemical structure and exhibited its pretreatment capability for fractionation and enzymatic hydrolysis of bagasse.