Cardiomyocytes are responsible for generating contractile force to pump blood throughout the body and are very sensitive to mechanical forces and can initiate mechano-electric coupling and mechano-chemo-transduction. Remarkable progress has been made in constructing heart tissue by engineered three-dimensional (3D) culture models and in recording the electrical signals of cardiomyocytes. However, it remains a severe challenge for real-time acquiring of the transient biochemical information in cardiomyocyte mechano-chemo-transduction. Herein, we reported a multifunctional platform by integrating a 3D stretchable electrochemical sensor with collagen hydrogel for the culture, electrical stimulation, and electrochemical monitoring of cardiomyocytes. The 3D stretchable electrochemical sensor was prepared by assembling functionalized conductive polymer PEDOT:PSS on an elastic scaffold, which showed excellent electrochemical sensing performance and stability under mechanical deformations. The integration of a 3D stretchable electrochemical sensor with collagen hydrogel provided an in vivo-like microenvironment for cardiomyocyte culture and promoted cell orientation via in situ electrical stimulation. Furthermore, this multifunctional platform allowed real-time monitoring of stretch-induced H2O2 release from cardiomyocytes under their normal and pathological conditions, as well as pharmacological interventions.