Mechanical stimulation can remodel the physiological mechanical microenvironment of cardiomyocytes and influence their maturation and injury progression. However, the dynamic process by which cardiomyocytes transition from adaptive maturation to injury remains unclear, because conventional endpoint assays cannot continuously track cellular states. Here, we developed a heart-on-a-chip platform that reconstructs physiological strain and fluidic microenvironments. The chip integrates traction force microscopy (TFM) and a cardiac troponin I (cTnI) biosensor, enabling synchronous monitoring of cardiomyocyte contractility and injury-associated phenotypes. Under physiological cyclic stretch (20% strain), the platform induced a mature cardiomyocyte phenotype, with the gap junction protein CX43 and cytoskeletal protein F-actin increasing by 1.22- and 2.03-fold, respectively. Through 14 days of dynamic mechano-chemical monitoring, we identified 25% strain as a critical turning point in cardiomyocyte mechanical responses. Below this threshold (5%-20%), mechanical stimulation promoted cellular maturation and enhanced contractile stress, whereas above this threshold (30%), it induced an injury-associated phenotype. This heart-on-a-chip provides a controllable and quantitative in vitro platform for optimizing mechanical stimulation windows, evaluating cardiomyocyte injury, and screening cardioprotective therapeutics.