Decellularized extracellular matrix has been suggested as a scaffold for heart valve tissue engineering or direct implantation. However, cell removal impairs the physical properties of the valve leaflet structure and the biomechanical properties of the valve leaflet. Matrix/polymer hybrid scaffold with improved biomechanical characteristics may be advantageous. Mesenchymal stem cells were obtained from rats. Porcine aortic valve leaflets were decellularized enzymatically and coated with biodegradable poly-4-hydroxybutyrate using an electrospinning technique, reseeded and cultured over a time period of 14 days. The morphologic, biochemical, and biomechanical characteristics of hybrid scaffolds were tested. Morphologic and biochemical assays indicated that mesenchymal stem cells survive and proliferate on hybrid scaffolds, and control decellularized scaffolds revealed comparable amounts of cell mass, 4-hydroxyproline and collagen after cultured in vitro for 14 days. Mechanical testing indicated hybrid scaffolds had superior tensile strength and elastic modulus. Altogether this study demonstrates the feasibility and improved biomechanical characteristics of a novel hybrid heart valve leaflet scaffold for an application in tissue engineering.