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Harnessing biomaterials for in vitro tissue construction has long been a research focus because of its powerful potentials in tissue engineering and pharmaceutical industry. Myocardium is a critical cardiac tissue with complex multiple muscular layers. Considering the specific characters of native cardiac tissues, it is necessary to design a biocompatible and biomimetic platform for cardiomyocyte culture and myocardium formation with sustained physiological function. In this study, we developed gelatin-based hydrogels chemically cross-linked by genipin, a biocompatible cross-linker, as cell culture scaffolds. Moreover, to achieve and maintain the functionality of myocardium, for instance, well-organized cardiomyocytes and synchronized contractile behavior, we fabricated gelatin-based hydrogels with patterned microstructure using a microcontact printing technique. Furthermore, graphene oxide (GO), with unprecedented physical and chemical properties, has also been incorporated into gelatin for culturing cardiomyocytes. Our results show that micropatterned genipin-cross-linked gelatin hydrogels are very helpful to promote alignment and maturation of neonatal rat ventricular cardiomyocytes. More interestingly, the presence of GO significantly enhances the functional performance of cardiomyocytes, including an increase in contraction amplitude and cardiac gene expression. The cultured cardiomyocytes reach a well-synchronized contraction within 48 h of cell seeding and keep beating for up to 3 months. Our study provides a new and easy-to-use gelatin-based scaffold for improving physiological function of engineered cardiac tissues, exhibiting promising applications in cardiac tissue engineering and drug screening.