Tissue engineering is a biotechnology that is used to develop biological substitutes to restore, maintain, or improve functions. Thus, the porous scaffolds are used to accommodate cells in tissue engineering. In this research, three dimensional (3D) bi-layered polyurethane (PU)-gelatin nanofibrous scaffolds were prepared by the electrospinning method, after which the capability of the released heparin as an anti-coagulation factor was evaluated. Electrospinning has been extensively investigated for the preparation of fibers that exhibit a high surface area to volume ratio. Results showed that scanning electron microscopy (SEM) micrographs exhibited a smooth surface as well as a highly porous and bead-free structure, in which fibers were distributed in the range of 100-600 nm. The modulus and ultimate tensile strength (UTS) decreased and increased, respectively, after crosslinking the reaction of polymers. This process also reduced swelling ratio, the hydrolytic biodegradation rate, and the release rate as a function of time. Moreover, an in vitro assay demonstrated that 3D nanofibrous scaffolds supported L929 fibroblast cell viability and that cells adhered and spread on the fibers. Based on the obtained results, the heparin-loaded electrospinning nanofibrous scaffolds have initial physicochemical and mechanical properties to protect neo-tissue formation.