Cellular mechanics plays an important role in physiological processes such as cell growth, differentiation, apoptosis and gene expression. Herein, we present a nano-imprinted structural color graphene film with anisotropic microgroove and two-dimensional (2D) photonic crystal structures for cardiomyocytes dynamic displaying. The anisotropic structural color graphene film was generated by a “sandwich” mold in one step with the assistance of two designed templates. Because of the microgroove structure and biocompatibility of the film, the cardiomyocytes could be induced into a highly ordered arrangement with recoverable autonomous beating. Meanwhile, the opposite surface of the film was imparted with defect-free 2D photonic crystal structures, giving it a vivid structural color and photonic band gap (PBG). We demonstrated that the flexible film would undergo synchronous volume or shape changes with the cardiomyocytes’ elongation and contraction during the beating process, showing cyclic changes of the structural color, which in turn could be converted into force. In addition, by integrating this anisotropic structural color graphene film with microfluidic channels, a novel and accurate heart-on-a-chip platform was established for real-time cell monitoring and drug screening. These characteristics of the present anisotropic structural color graphene films make them extremely valuable in the field of biomedicine.