Abstract We provide a blend of cardiac decellularized extracellular matrix (dECM) from porcine ventricular tissue and fibrinogen for the formation of an in-vitro 3D cardiac cell culture model. Rapid and specific coagulation with thrombin allows gentle inclusion of cells while avoiding sedimentation during formation of the dECM-fibrin composite. We use the system in co-culture with Nor-10 fibroblasts to enhance cardiogenic differentiation of the H9c2 myoblast cell line. The combination of co-culture and appropriate substrate allows to abrogate the use of retinoids, classically considered necessary for cardiogenic H9c2 differentiation. Further enhancement of differentiation efficiency is obtained by 3D embedding. We then proceed with culture of rat neonatal cardiomyocytes in the 3D system. While for H9c2 cells, the collagen content of the dECM was the key factor required for efficient differentiation, the use of dECM-fibrin has specific advantages regarding the culture of neonatal cardiomyocytes. Calcium imaging and analysis of beating motion both indicate that the dECM-fibrin composite significantly enhances recovery, frequency, synchrony and maintenance of spontaneous beating, as compared to various controls including matrigel, pure fibrin and collagen I, but also a fibrin-collagen I blend.