Electrospun nanofiber is one of the promising alternatives for use in tissue engineering and drug delivery due to its controllable characteristics. However, choosing an appropriate biomaterial for a specific tissue regeneration plays a significant role in fabricating functional tissue-engineered constructs. Heart extracellular matrix (ECM)-derived electrospun nanofiber which mimic the physicochemical and structural characteristics of cardiac tissue is advantageous for cardiac tissue engineering. In this study, acellular calf heart ECM has been investigated as a potential biomaterial to be electrospun in a novel combination with poly vinyl pyrrolidone (PVP), gelatin (Gel) and polycaprolactone (PCL) for cardiac tissue engineering. The obtained fibers were aligned, uniform, and bead free. After fabrication, the scaffolds were cross-linked in glutaraldehyde vapor to become mechanically stronger and dissoluble in the aqueous environments. Considering surface topography, biocompatibility, hydrophilicity, and mechanical properties, the fabricated hybrid electrospun ECM/PVP/Gel/PCL fibers can be proposed as a biomimetic scaffold for heart tissue engineering applications.