The spider silk protein bilayer vascular scaffold (SSPBVS) based on RGD-recombinant spider silk protein (pNSRl6), gelatin (Gt), chitosan (CS), and polycaprolactone (PCL) was prepared using electrospinning method. To evaluate the biological safety of SSPBVS, which has potential application in tissue engineering blood vessel, acute toxicity test, skin irritation test, skin sensitization test, single cell gel electrophoresis test, pyrogeneous test, chromosomal aberration test, and micronucleus test were carried. The results demonstrated that distribution of scaffold fiber was dense, with high porosity and no adhesion phenomenon was found. When Mesenchymal stem cells (MSCs) were cultured with vascular scaffold extract, they proliferated rapidly with fusiform morphology. Statistics showed that there was no significant difference in rat organ index between SSPBVS extract group and control group. SSPBVS extract only had slight irritation on the standard deviation (SD) rat skin, and no sensitization was found. SSPBVS extract did not initiate DNA damage of MSCs, and nuclear DNA was complete. Body temperature elevation value of SD rat in SSPBVS extract group was less than 0.2 degrees C. The SSPBVS extract did not cause gene mutation of the chromosome. PCE ratio of SSPBVS extract group was 3.1 +/- 1.5 parts per thousand. This study suggested that SSPBVS did not bring significant toxic effect on MSCs in vitro and had potential as scaffold in tissue engineering blood vessel.