A promising approach in the selective separation and modification of cellulose from raw biomass under a mild alkali process was proposed. In our study, ball milling was applied to wheat straw prior to alkali treatment. With ball milling, ultrafine powder formed an amorphous microstructure and displayed a level of solubilization in aqueous NaOH higher than that of general ground samples. Alkali-treated ultrafine powder resulted in up to 93.76% removal of hemicellulose and 86.14% removal of lignin, whereas cellulose remains largely undissolved. A high glucose yield (98.48%) was obtained via a 72 h enzymatic hydrolysis. X-ray diffraction and solid state ¹³C cross-polarization magic angle spinning nuclear magnetic resonance analysis revealed evidence of the transformation of crystalline cellulose I to cellulose II in alkali-treated ultrafine wheat straw. Prolonging the alkaline treatment time can significantly decrease the level of cellulose hydrogen bonding and increase the hydrolysis yield. The combination of ultrafine ball milling and low-severity alkali treatment played a significant role in the cellulose supramolecular change, which can then be used for downstream biorefinery processes or as a feedstock for the biomaterial industry.