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Rapid formation of a continuous endothelial cell (EC) monolayer with healthy endothelium function on the luminal surface of vascular implants is imperative to improve the longtime patency of small-diameter vascular implants. In the present study, we combined the contact guidance effects of aligned nanofibers, which enhance EC adhesion and proliferation because of its similar fiber scale with native vascular basement membranes, and aligned microfibers, which could induce EC elongation effectively and allow ECs infiltration. It was followed by successive immobilization of collagen IV and laminin to fabricate a biomimetic basement membrane (BBM) with structural and compositional biomimicry. The hemolysis assay and platelet adhesion results showed that the BBM exhibited excellent hemocompatibility. Meanwhile, the adhered human umbilical vein endothelial cells (HUVECs) onto the BBM aligned along the orientation of the microfibers with an elongated morphology, and the data demonstrated that the BBM showed favorable effects on EC attachment, proliferation, and viability. The oriented EC monolayer formed on the BBM exhibited improved antithrombotic capability as indicated by higher production of nitric oxide and prostacyclin (PGI2). Furthermore, fluorescence images indicated that HUVECs could infiltrate into the BBM, implying the BBM's ability to enhance transmural endothelialization. Hence, the BBM possessed the properties to regulate EC behaviors and allow transmural ingrowth, demonstrating the potential to be applied as the luminal surface of small-diameter vascular implants for rapid endothelialization.