Chirality and repulsion interactions among sulfate cellulose nanocrystals (CNCs) have vital impact on the formation of a cholesteric liquid crystal (CLC) phase in a suspension or solidified film. In this work, a facile sonication treatment was applied to change the structure and repulsion interactions of CNCs and consequently tune the chiroptical properties of the resultant films. The results show that increasing the sonication energy either by improving the input power or prolonging the aging time resulted in the reduction of particle size and surface charge density, thereby increasing the cholesteric pitch and red-shifting the reflective wavelength of the iridescent films. The optical properties of the film followed the regulation of Bragg reflection and thin-film interference. However, an over-energy input would result in the multi-dispersion of the CNCs according to the level of the surface charge density, thus leading to the formation of polydomain CLC instead of planar CLC because of multi-distributed intra-axial drive forces. Hence, a schematic model was built up to describe the structure transition, as well as the color variation and to correlate the mesoscopic behavior of CNCs and the microscopic interactions of electrostatic repulsions, hydrogen bonding affinity and chirality. Hence, we provide some meaningful information on building up a hierarchical organization assembled from charged rigid biological rods, and help to recognize the structure–color mechanism of solidified films of polysaccharide nanocrystals.