The demands for strong integrated biopolymer materials have substantially increased across various industries. In this study, a biomimic strategy was proposed to prepare the poly(dopamine)‐functionalized carbon nanotubes (PDCNTs) via mussel‐inspired chemistry. The graphene dispersion was prepared in aqueous bovine serum albumin solution by ultrasonic treatment through a facile and green approach. Inspired by the excellent integration of mechanical properties and hierarchical nano/microscale structure of natural nacre, we fabricated soy isolate protein (SPI)‐based nanocomposite film with 2D graphene nanosheets and 1D surface‐functionalized PDCNTs through a layer‐by‐layer assembly process. The morphology and thickness of graphene nanosheets were analyzed by atomic force microscopy. The successful surface modification of PDCNTs was confirmed by X‐ray photoelectron spectroscopy and transmission electron microscopy. The cross‐linking hierarchical structure of the SPI hybrid film was observed in scanning electron microscopy images. A combination of multiple interfacial interactions and enhanced adhesion between the PDCNTs‐graphene conjugation and SPI matrix resulted in a remarkable improvement in the mechanical properties of the SPI‐based nanocomposites. When compared with the unmodified film, the tensile strength and elongation at break of the hybrid film were simultaneously increased by 158.93 and 78.67%, respectively. As a result of the enhanced tortuosity effect, the water vapor permeability was significantly reduced by 33.65%. In addition, the resultant film also possessed favorable water resistance, thermal stability, and ultraviolet–visible light barrier behavior. This work provided a novel bio‐inspired interfacial toughening strategy for constructing high performance biopolymer nanocomposites. POLYM. COMPOS., 40:E1649–E1661, 2019. © 2018 Society of Plastics Engineers