The studies investigated the effect of the lignocellulosic composition of different segments of Reutealis trisperma waste on nanocrystalline cellulose (NCs) characteristics. The concentration of lignin, hemicellulose, and cellulose in Reutealis trisperma waste defines the characteristics of the isolated NCs. The mesocarp peel (RP) and endocarp shell (RS) from Reutealis trisperma fruit have approximately similar cellulose content at ∼29–31%, but different lignin and hemicellulose concentrations. Alkalization and bleaching produced cellulose and hemicellulose mixtures. A high concentration of hemicellulose in RP-cellulose caused efficient dissolution during the hydrolysis process, producing NCs with smaller morphology. Thermal stability, zeta potential, and colloidal suspension size are also varied for NC-RP and NC-RS due to the different sizes of NCs. The NCs produced from RP have higher crystallinity, particle size, and thermal stability than NCs from RS. This study revealed that different characteristics of NCs generated from different segments of the Reutealis trisperma biowaste might be related to the ability of hemicellulose to facilitate hydrolysis into smaller NCs fragments.