Cellulose nanocrystals (CNCs) are inherently right-handed nanostructures that originate from nature, showing chirality in their fibrils, bundles, and self-assembled films. However, the enantio-specific interaction between CNCs and other chiral molecules has not been explored so far. In this study, we first demonstrated a chirality-related difference in the composite films of cellulose nanocrystals and histidine with a D- or L-configuration. The distinction is not only presented in the self-assembled nanostructures of CNCs, optical properties, and the thermal decomposition of composites but also in the crystallization of the amino acid. We suppose that it might have originated from the packing of amino acids on the twisted surface of CNCs. The knowledge about the enantio-specific interaction between the chiral amino acid and polysaccharide nanostructure is of significant importance for developing a new strategy for enantiomeric separation.