Layer-by-layer self-assembly (LBL) is an effective method to prepare potential biomaterial with multilayer coatings, and few reports have focused on the variation of oriented microstructure during LBL process. In this study, polycaprolactone (PCL) and type І collagen (COL) were electrospun to oriented nanofibrous mats, and chitosan (CS) and COL molecules were then deposited on the mats by LBL technique. Zeta potential, FT-IR analysis and XPS measurement indicated the successful fabrication and modification. Changes in surface morphology and increase in surface roughness were observed in LBL process. Additionally, LBL-structured mats exhibited improved mechanical properties with the maximal tensile strength of 35.1 ± 7.0 MPa and the best elongation of 106.0 ± 11.5 %. CCK-8 and live/dead assays illustrated that the cell viability of the mats increased more than 20 % after LBL modification. More importantly, cells seeded onto the mats showed oriented adhesion and growth along the direction of nanofiber arrangement in LBL modified mats, which provided an effective strategy for realizing the controlled growth of cells.