Abstract: The composite films based on collagen (Col) reinforced by dialdehyde cellulose nanocrystals (DACs) were fabricated for wound dressing. Cellulose nanocrystals (CNCs) were obtained by acid hydrolysis. The surface of CNCs was treated by sodium periodate to produce DACs with different degrees of oxidation (D_O = 8.6%—51.3%). The length and height of DACs were (208 ± 30) nm and (9 ± 3) nm, respectively. The Col/DACs-IBP multilayer films were prepared by alternately filtering DACs-Ibuprofen (IBP) solution and Col solution. The structure and property of the films were characterized by scanning electron microscopy, electronic universal testing machine, and UV-Vis spectrophotometer. The aldehyde groups of DACs reacted with the amino groups of Col, resulting in chemical cross-linking networks between the adjacent layers. Therefore, increasing D_O of DACs and/or improving the dispersity of DACs and Col could enhance the covalent interactions between the adjacent layers, by which tight and uniform structure of films were formed. The Col/DACs-IBP multilayer films showed outstanding mechanical properties and light transmittance. The optimal tensile strength was 54.2 MPa, which was improved by 152.1%. The optimal light transmittance was up to 95.7%, which was improved by 13.5%. The water holding capacity of the films was reduced upon increasing D_O of DACs. in vitro Drug release experiments suggested that the IBP loaded in films was slowly released up to 89.0% within 24 h. Furthermore, the films could obviously promote the adhesion and proliferation of cells within the test time.