<p>As one of the most popular polysaccharide derivatives with high chiral recognition ability, cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) has been successfully applied in the enantioseparation of a wide range of racemates for various purposes. However, the derivative is usually synthesized with an amorphous structure, and its effect on enantioseparation ability remains obscure. To obtain a better understanding of the correlation between its structure and enantioseparation ability, in this study, CDMPC derivatives with crystalline and amorphous structures were prepared through carbamoylation followed by anti-solvent precipitation. Their chiral recognition abilities were evaluated using high-performance liquid chromatography (HPLC) with chiral stationary phases (CSPs). Interestingly, the ordered arrangement of the helical polymer chains of CDMPC was effectively induced by the aromatic solvent after derivatization, forming a crystalline derivative (<b>c-CDMPC</b>) with a uniform and compact worm-like morphology, whereas the amorphous derivative (<b>a-CDMPC</b>) was obtained by regular treatment with a polar alcoholic solvent. The <b>c-CDMPC</b>–based CSP exhibited higher chiral recognition abilities with much shorter retention times for most racemates in this study, compared to the <b>a-CDMPC</b>–based CSP. In particular, flavanone (<b>Rac-7</b>), an important chiral drug with anti-inflammatory, anti-tumor, and cardiovascular protective activities, was even better resolved with higher enantioselectivity on crystalline <b>c-CDMPC</b> than on Chiralcel OD, a commercialized chiral column based on CDMPC. This indicates that the crystalline ordered arrangement played a critical role in the chiral recognition ability of the cellulose derivative.</p>