Cellulose nanocrystals (CNCs) are perfect rodlike nanofibers that can self-assemble and form a chiral nematic phase. We found that different self-assembling morphologies could be formed by different size-unified CNCs. This study reported a facile and new approach of fractionating raw (unseparated) CNCs in a wide particle size distribution (9-1700 nm) into a series of narrower size ranges to obtain size-unified CNCs via a well-designed multistage separation process composed of layered filter membranes with different pore size cutoffs followed by a fast pressurized filtration. The smaller size-unified CNCs readily self-assembled into polish chiral nematic phases with larger pitch value as compared to larger size-unified CNCs. Such a distinction among different chiral nematic phases and pitch values as functions of size was addressed by a mathematical evaluation, which suggested that the reduced volume fraction of the anisotropic phase as a function of both increased ionic strength and reduced crystallinity of rigid-rod-like CNCs is a critical factor. In addition, Fourier-transform infrared spectroscopy, thermogravimetric analysis, and X-ray diffraction results revealed that different size-unified CNCs exhibited particular thermal stabilities and crystallinities even though their chemical and crystalline structures remained unchanged. The discrepancies in physicochemical characteristics and self-assembling chiral nematic behavior among different size-unified CNCs may benefit the specific functionalization of cellulose materials using size-unified fibers instead of raw CNCs containing mixed small and large fibers.