Establishing a long-term and multi-parameter cell physiological assessment platform is of great significance for drug screening and cardiology research. Invasive and label-based recording platform usually destruct cell viability, hinder the long-term chronic monitoring of cardiomyocytes. Here, we develop a multifunctional cardiomyocyte-based biosensor, which integrates microelectrodes and interdigitated electrodes for noninvasive simultaneous monitoring of cardiomyocyte electrophysiology, mechanical beating and growth viability. Through further analysis of the typical and chronic physiological signals, it is demonstrated that the cardiomyocytes possess a stable and mature state after 192 h culture, which is applicable to the pharmaceutical analysis. It is believed that the multifunctional biosensor will provide a practical platform for the study of cardiac physiology/pathology, and be widely used in the biomedical field.