Bionic grafts hold great promise for directing tissue regeneration. In vascular tissue engineering, although a large number of synthetic grafts have been constructed, these substitutes only partially recapitulated the tri-layered structure of native arteries. Synthetic polymers such as poly(l-lactide-co-ε-caprolactone) (PLCL) possess good biocompatibility, controllable degradation, remarkable processability, and sufficient mechanical strength. These properties of PLCL show great promise for fabricating synthetic vascular substitutes. Here, tri-layered PLCL vascular grafts (TVGs) composed of a smooth inner layer, circumferentially aligned fibrous middle layer, and randomly distributed fibrous outer layer were prepared by sequentially using ink printing, wet spinning, and electrospinning techniques. TVGs possessed kink resistance and sufficient mechanical properties (tensile strength, elastic modulus, suture retention strength, and burst pressure) equivalent to the gold standard conduits of clinical application, i.e., human saphenous veins and human internal mammary arteries. The stratified structure of TVGs exhibited a visible guiding effect on specific vascular cells including enhancing endothelial cell (EC) monolayer formation, favoring vascular smooth muscle cells’ (VSMCs) arrangement and elongation, and facilitating fibroblasts’ proliferation and junction establishment. Our research provides a new avenue for designing synthetic vascular grafts with polymers.