The cell-substrate distance is a direct indicator of cell adhesion to extracellular matrix which is indispensable in cell culture. A real-time monitoring approach can provide a detailed profile of cell adhesion, so that enables the detecting of adhesion-related cell behavior. In this work, we report a novel real-time impedance-based method to record the adhesion profile of cardiomyocyte, overcoming its inscrutability due to the primary culture. Microfabricated biosensors are applied in cardiomyocyte culture after characterizing the cell-free system. Cyclic frequency scanning data of cell-related impedance are generated and automatically fit into the equivalent circuit model, which is established using electrochemical impedance spectroscopy. The data are displayed as the alteration of normalized cell-substrate distance and the essential parameters for manual electric cell-substrate impedance sensing calibration of absolute distance. The time course displays a significant decline in the equivalent cell-substrate distance, from 155.8 to 60.2 nm in the first 20 h of cardiomyocyte culture. Furthermore, the cardiomyocytes cultured in long-term medium and short-term medium (ACCT) for 10 h exhibit distinct difference in adhesion rate as well as cell-substrate distance (72 vs 68 nm).