Objective To prepare a bilayer spider silk protein vascular scaffold using electrospinning, observe microstructure of the vascular scaffold and study its biomechanical properties and cell compatibility. Methods Spinning solution was electrospun to prepare (pNSR16/PCL/CS)/(pNSR16/PCL/Gt) bilayer spider silk protein vascular scaffold using rotating receiving rod as the collection device. The effects of mass fraction and wall thickness on the porosity, bursting strength, tensile properties, suture retention strength and water permeability of the vascular scaffold were investigated, and cytotoxicity and cell adhesion property of the vascular scaffold were tested. Results The vascular scaffold presented three-dimensional porous microstructure with randomly distributed fibers. The bursting strength, tensile strength and suture retention strength were directly proportional to mass fraction and wall thickness, but the porosity, water permeability and elongation at break were inversely proportional to mass fraction and wall thickness. The bursting strength range of vascular scaffold was 43~183 kPa, which was higher than the physiological blood pressure; the suture strength was above 0.19 N, which was consistent with the transplantation requirement in vivo; the tensile strength was higher than that of human radial artery, which met the transplantation requirement in vivo; the range of water permeability was 0.3~0.6 mL?min-1?cm-2. The vascular scaffold had no cytotoxicity and facilitated the adhesion and proliferation of endothelial cells. Conclusions It is feasible to prepare the bilayer spider silk protein vascular scaffold through electrospinning. The superior biomechanical properties and biocompatibility properties show that the bilayer spider silk protein vascular can be used for construction of the tissue engineered blood vessels in vitro, with prospect for further vascular graft study, which lays a foundation for its clinical application.