An efficient and sustainable pretreatment, such as organosolv pretreatment that produces high-quality lignin and highly digestible carbohydrates, could enable the potential complete utilization of lignocellulosic biomass. Demand for bio-based solvents with a high boiling point, low viscosity, and negligible toxicity is increasing. Herein, we report the use of dimethyl isosorbide (DMI) as a solvent to fractionate lignocellulosic biomass into its main components for the first time. High lignin removal efficiency (91.2%) with good cellulose retention (around 80%) could be achieved during the pretreatment of Eucalyptus by DMI/H2O co-solvents under a mild condition. A near-complete cellulose conversion to its monosaccharide could be realized at a relatively low enzyme loading of 20 FPU g−1 glucan. The addition of water could suppress the condensation of lignin, yielding high-quality lignin with a good fraction of β-O-4 linkages reserved (24.8%) and homogeneous molecular weight (Đ<2) suitable for depolymerization to mono-aromatic chemicals. Besides its highly digestible nature, the high quality of the cellulose-rich residue is also demonstrated from a material perspective. A more efficient fibrillation of obtained pulp to nanocellulose was developed, leading to a promising potential of energy saving compared to the traditional bleaching pathway. Overall, this work developed a mild pretreatment technology as a potential basis for a green and closed-loop biorefinery concept for converting lignocellulosic biomass to multiple products (high-quality lignin, fermentable sugars, or functional materials).