In this study, recombinant spider silk protein (pNSR16) was blended with polycaprolactone (PCL), gelatin (Gt), and chitosan (CS) to prepare (pNSR16/PCL/CS)/(pNSR16/PCL/Gt) bilayer small-diameter vascular scaffold through electrospinning in order to imitate multilayer structure of natural vessel. The surface morphology and properties were characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, water contact angle, and mechanical tensile testing. The blood compatibility of scaffold was evaluated by recalcification coagulation time. Mesenchymal stem cells (MSC) were separated from SD rat bone marrow, and MTT assay was used to evaluate cell growth. Cell compatibility and interaction mechanism between bilayered vascular scaffold and MSC were studied. Subcutaneous implantation was performed to evaluate in vivo inflammatory reaction and degradation behavior. The preliminary results indicated that the scaffold fibers were relatively uniform, and hydrophilic performance and blood compatibility were good. The values of the tensile strength and elongation at break of vascular scaffold were 39 +/- 2MPa and 118 +/- 4%, respectively. MSC morphology was uniform and spindle, whose steady growth period was reached on the fifth day. MSC adhered well and proliferated vigorously on vascular scaffold surface. A large number of MSC could migrate into (pNSR16/PCL/CS)/(pNSR16/PCL/Gt) scaffold interior. pNSR16 and Notch receptor had interaction function at protein level. The preparation of (pNSR16/PCL/CS)/(pNSR16/PCL/Gt) laid foundation for the construction of tissue engineering vessel.