A facile, inexpensive, and green approach for the production of stable graphene dispersion was proposed in this study. We fabricated soy protein isolate (SPI)-based nanocomposite films with the combination of 2D negative charged graphene and 1D positive charged polyethyleneimine (PEI)-modified cellulose nanocrystals (CNC) via a layer-by-layer assembly method. The morphologies and surface charges of graphene sheets and CNC segments were characterized by atomic force microscopy and Zeta potential measurements. The hydrogen bonds and multiple interface interactions between the filler and SPI matrix were analyzed by Attenuated Total Reflectance-Fourier Transform Infrared spectra and X-ray diffraction patterns. Scanning electron microscopy demonstrated the cross-linked and laminated structures in the fracture surface of the films. In comparison with the unmodified SPI film, the tensile strength and surface contact angles of the SPI/graphene/PEI-CNC film were significantly improved, by 99.73% and 37.13% respectively. The UV-visible light barrier ability, water resistance, and thermal stability were also obviously enhanced. With these improved functional properties, this novel bio-nanocomposite film showed considerable potential for application for food packaging materials.