In this paper, a novel combination method of electrospinning and rapid prototyping (RP) fused deposition modeling (FDM) is proposed for the fabrication of a tissue engineering heart valve (TEHV) scaffold. The scaffold preparation consisted of two steps: tri-leaflet scaffold fabrication and heart valve ring fabrication. With the purpose of mimicking the anisotropic mechanical properties of the natural heart valve leaflet, electrospun thermoplastic polyurethane (ES-TPU) was introduced as the tri-leaflet scaffold material. ES-TPU scaffolds can be fabricated to have a well-aligned fiber network, which is important for applications involving mechanically anisotropic soft tissues. We developed ES-TPU scaffolds as heart valve leaflet materials under variable speed conditions and measured fiber alignment by fast Fourier transform (FFT). By using FFT to assign relative alignment values to an electrospun matrix, it is possible to systematically evaluate how different processing variables affect the structure and material properties of a scaffold. TPU was suspended at certain concentrations and electrospun from 1,1,1,3,3,3-hexafluoro-2-propanol onto rotating mandrels (200―3000 rpm). The scaffold morphological property and mechanical anisotropic property are discussed in the paper as a function of fiber diameter and mandrel RPM. The induction of varying degrees of anisotropy imparted distinctive material properties to the electrospun scaffolds. A dynamic optimum design of the heart valve ring graft was constructed by FDM. Fabrication of a 3D heart valve ring was constructed using pro-engineer based on optimum hemodynamic analysis and was converted to an STL file format. The model was then created from PCL which was sewed and glued with electrospun nanofibrous leaflets. This proposed method was proven as a promising fabrication process in fabricating a specially designed graft with the correct physical and mechanical properties.