A bilayered scaffold was fabricated via electrospinning layer-by-layer with the inner layer composed of blend fibers of spider silk protein and gelatin (pNSR16/Gt), then the polyurethane (PU) layer on the outside. The physicochemical and biological performance of scaffold were investigated. The pNSR16/Gt-PU composite nanofibers with interconnected pores was detected a porosity of 88.7 +/- 1.5%; with mechanical properties, including an appropriate permeability of 6.8 +/- 0.2 ml min(-1) cm(-2), breaking strength (24.6 +/- 3.6 MPa) and elasticity up to 145 +/- 3.8% strain, and burst pressure reached 276 +/- 7.1 kPa as well as an accepted suture retention strength (4.9 +/- 0.8 N), optimized to mimic the nature artery. The pNSR16/Gt-PU bilayered scaffold also proved to be capable to support cell growth and proliferation of Sprague-Dawley rat aortic endothelial cells by 242 +/- 10% (7 days vs. control). After all, the intimal surface promotes endothelialization and being highly anti-thrombogenic, while the mismatch in mechanical properties outside induces intimal hyperplasia, it is desirable to develop a bioactive material with improved mechanical properties for small-diameter vascular graft. (C) 2014 Society of Plastics Engineers