A comprehensive elucidation of the unexpected adverse events that occur in skeletal myoblast transplantation is fundamental for the optimization of myocardial therapeutic effects. However, a well-defined method to study the interactions between skeletal myoblasts and cardiomyocytes during the healing process is out of reach. Here, we describe a microfluidic method for monitoring the interactions between skeletal myoblasts and hypoxia-injured cardiomyocytes in a spatiotemporally-controlled manner, mimicking the in vivo cell transplantation process. A myocardial hypoxia environment was created using an oxygen consumption blocking reagent, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone. Meanwhile, the interactions between the skeletal L6 myoblasts and hypoxia-injured myocardium H9c2 cells were investigated, and the effects of a L6 conditional medium on H9c2 cells were comparatively analyzed by quantitatively measuring the morphological and pathophysiological dynamics of H9c2 cells. The results showed that skeletal myoblasts could repair hypoxia-injured H9c2 cells mainly through direct cell-to-cell interactions. This simple on-chip assay for investigating myocardial repair processes may provide avenues for the in vitro screening of drug-induced cardiotoxicity.