Objective To prepare a spider silk protein bilayer small diameter vascular scaffold using electrospinning, and to observe the blood compatibility in vitro. Methods The Arg-Gly-Asp-recombinant spider silk protein (pNSR16), polycaprolactone (PCL), gelatin (Gt), and heparin (Hep) were blended. Spider silk protein bilayer small diameter vascular scaffold (experimental group) was prepared by electrospinning, with pNSR16 ∶ PCL ∶ Hep (5 ∶ 85 ∶ 10, W/W) hybrid electrospun solution as inner spinning solution and pNSR16 ∶ PCL ∶ Gt (5 ∶ 85 ∶ 10, W/W) hybrid electrospun solution as outer spinning solution, but pNSR16 ∶ PCL (5 ∶ 85, W/W) hybrid electrospun solution was used as inner spinning solution in control group. The scaffold structure of experimental group was observed under scanning electron microscope (SEM); and the hemolysis rate, recalcification clotting time, dynamic clotting time, platelet adhesion, and platelet activation in vitro were compared between 2 groups. Results SEM results showed that bilayer fibers of scaffold were quite different in experimental group; the diameter distribution of inner layer fibers was relatively uniform with small pores, however diameter difference of the outer layer fiber was relatively big with big pores. The contact angle, hemolysis rate, recalcification clotting time, and P-selectin expression of scaffold were (35 ± 3) ° , 1.2% ± 0.1%, (340 ± 11) s, and 0.412 ± 0.027 respectively in experimental group, and were (70 ± 4) ° , 1.9% ± 0.1%, (260 ± 16) s, and 0.678 ± 0.031 respectively in control group; significant difference were found in indexes between 2 groups (P lt; 0.05). With the extension of time, the curve of coagulation time in experimental group sloped downward slowly and had a long time; the blood clotting index values before 30 minutes were significantly higher than those in control group (P lt; 0.05). Platelet adhesion test showed that the scaffold surface almost had no platelet adhesion in experimental group. Conclusion The spider silk protein bilayer small diameter vascular scaffold could be prepared through electrospinning, and it has good blood compatibility in vitro.