In this study, we propose a complete biomass fractionation strategy where all three major biopolymers, namely, cellulose, hemicellulose, and lignin, are separated with higher efficiency and purity. Sequential treatment of hybrid poplar wood using autohydrolysis (160 degrees C,60 min) and 1-ethyl-3-methylimidazolium acetate activation (60 degrees C,3 h) resulted in significantly improved enzymatic saccharification and fractionated 85% cellulose and 67% hemicellulose. The resulting solid fraction contained 90% (w/w) lignin, which was equal to 71% yield based on the original biomass composition. The proposed two-step pretreatment process improved lignin yield by 77% and 23% compared to the single-stage ionic liquid activation or autohydrolysis, respectively. Structural characterization by 2D nuclear magnetic resonance spectroscopy and small-angle neutron scattering revealed that the isolated lignin sustained minimal modifications to interunit linkages and exhibited high thermotolerance as well as unique functionality, thereby highlighting the benefits of this process for lignin fractionation.