While electroporation has been widely used as a physical method for gene transfection in vitro and in vivo, its application in gene therapy of cardiovascular cells remains challenging. Due to the high concentration of ion-transport proteins in the sarcolemma, conventional electroporation of primary cardiomyocytes tends to cause ion-channel activation and abnormal ion flux, resulting in low transfection efficiency and high mortality. In this work, a high-throughput nanoelectroporation technique based on a nanochannel array platform is reported, which enables massively parallel delivery of genetic cargo (microRNA, plasmids) into mouse primary caidiomyocytes in a controllable, highly efficient, and benign manner. A simple "dipping-trap "approach was implemented to precisely position a large number of cells on the nanoelectroporation platform. With dosage control, our device precisely titrates the level of miR-29, a potential therapeutic agent for cardiac fibrosis, and determines the minimum concentration of miR-29 causing side effects in mouse primary cardiomyocytes. Moreover, the dose-dependent effect of miR-29 on mitochondrial potential and homeostasis is monitored. Altogether, our nanochannel array platform provides efficient trapping and transfection of primary mouse cardiomyocyte, which can improve the quality control for future microRNA therapy in heart diseases.