In this study, crystalline cellulose was prepared through hydrolysis of jute fiber and was used as reinforcement of gelatin-based biocomposite film. The effects of crystalline celluloses loading on the morphology, mechanical properties and water sensitivity of the biocomposite were investigated by means of Scanning electron microscopy, tensile strength testing and water absorption testing. The developed biocomposite film showed homogeneous dispersion of crystalline celluloses within the gelatin matrix and strong interfacial adherence between matrix and reinforcement. A significant increase in tensile strength and E Modulus was also found (tensile strength was 25.4 MPa for pure gelatin and 48.2 MPa for 2% crystalline celluloses/gelatin film at 45% relative humidity), which was further induced by gamma radiation. The resulting biocomposite film also showed a higher water resistance and excellent biocompatibility. Therefore, crystalline celluloses played an important role in improving the mechanical properties as well as water resistance of the biocomposite film.