The present study aimed to develop a new bio-nanocomposite film based on gum arabic (GA) reinforced with cellulose nanocrystals (CNC). CNC was successfully fabricated and its microstructure was characterized. Subsequently, the effects of CNC on the rheological, physicochemical and functional properties of GA-based films were systematically evaluated. Results showed that the tensile strength (2.21 MPa) and elongation at break (62.79%) of film incorporated with 4% (w/w) CNC were effectively increased compared with the GA film (1.08 MPa and 42.50%). Additionally, 4% CNC reduced the water vapor and oxygen permeability by 10.61% and 25.30% respectively, while improved the ultraviolet light barrier and thermal stability of film. The well dispersion and filling effect of nanofiller contributed to form a compact and homogeneous film structure. Furthermore, the film containing 4% CNC decreased the weight loss of strawberries by 23.80% compared with the control group, thus delaying the deterioration of strawberry quality during storage.