The work aims to explore the suitable process conditions and properties change rule of cellulose nanocrystals (CNC) films prepared from 3 different raw materials (eucalyptus bleached kraft pulp, long-staple cotton and short-staple cotton), so as to provide reference for the study of CNC structural color anti-counterfeiting films. CNC suspensions were prepared by sulfuric acid hydrolysis of 3 different raw materials under appropriate hydrolysis conditions, and the particle size, Zeta potential and microscopic morphology of CNC particles were measured. CNC films were formed by evaporation drying self-assembly method. The chemical structure, crystal type, microstructure, mechanical properties, polarizing properties and maximum reflection wavelength of the films were measured. The CNC suspensions prepared from 3 different raw materials had good stability and retained the unique functional groups of cellulose and natural Iβ crystal form. With the increase of acid hydrolysis time, the particle size of CNC gradually decreased, and the length-diameter ratio was about 15. The crystallinity of CNC prepared from eucalyptus bleached kraft pulp was lower than that of CNC prepared from short-staple cotton and long-staple cotton, and the flexibility and tensile strength of the prepared CNC film were better than those of CNC film made from short-staple cotton and long-staple cotton. The cross sections of CNC films prepared from the 3 raw materials all had cholesteric liquid crystal layered structure and birefringence effect under polarized light microscope. With the increase of acid hydrolysis time, the pitch of CNC films gradually decreased, the maximum reflection wavelength also gradually reduced, and the color of the films all had blue-shifted. Compared with the CNC films prepared from short-staple cotton and long-staple cotton, the birefringence of the CNC films prepared from eucalyptus bleached kraft pulp was weaker. The type of raw materials and hydrolysis time both have an effect on the particle size, crystallinity and birefringence of the prepared CNC. With the increase of acid hydrolysis time, the pitch of CNC film gradually decreases, which can adjust the reflection wavelength of cellulose nanocrystalline film in a certain range, so as to adjust the structural color of cellulose nanocrystalline film. This cellulose nanocrystalline material is expected to be used in the field of anti-counterfeiting packaging in the later stage.