The complexity of cardiovascular disease requires that the materials for preparing vascular grafts possess good biocompatibility, high mechanical property, and even some excellent additional properties. In this study, polycaprolactone (PCL) with good mechanical property and natural source silk fibroin (SF) were electrospun into PCL/SF nanofibers to obtain the nanofibrous substrate. With the addition of SF, the mechanical property of PCL/SF nanofibrous mats was maintained to a certain extent. While, the hydrophilicity of PCL/SF nanofibrous mats was greatly improved which is more suitable for immersive layer-by-layer assembly (LBL). The oppositely charged heparin (Hep) and chitosan (CS) were alternatively deposited on the surface of PCL/SF nanofibers via LBL. After implanting human umbilical vein endothelial cells (HUVEC) on the LBL-structured nanofibrous mats for 48 h, it was confirmed that the CS/Hep bilayers enhanced the biocompatibility of the nanofibers. Furthermore, the results of the antibacterial test showed that the antibacterial effects of the LBL-structured nanofibrous mats for Escherichia coli and Staphylococcus aureus were both achieved 95% when the number of Hep/CS bilayer was 10. It can be demonstrated that the LBL-structured nanofibrous mats with improved biocompatibility and reduced infectivity had been prepared successfully, and can be potentially used in vascular grafts. (C) 2020 Published by Elsevier B.V.