Reconstitution of urethral defects through a tissue-engineered autologous urethra is an exciting area of clinical urology research. Despite rapid advances in this field, a tissue-engineered urethra is still inaccessible to clinical applications because of the poor vascularization of the current scaffold materials, especially for the reconstruction of complex urethral defects. In this study, we report the preparation of multifaceted bio-interfacing tissue-engineered autologous scaffolds based on alternating block polyurethane (abbreviated as PU-alt), a kind of tubular scaffold with a hierarchical nanofiber architecture, flexible mechanical properties and a hydrophilic PEGylation interface capable of promoting adhesion, oriented elongation, and proliferation of New Zealand rabbit autologous urethral epithelial cells (ECs) and smooth muscle cells (SMCs) simultaneously, and also upregulating the expression of keratin (AE1/AE3) in ECs and contractile protein (α-SMA) in SMCs as well as the subsequent synthesis of elastin. Three months in vivo scaffold substitution of rabbit urethras displayed that the engineered autologous PU-alt scaffold grafts, with a coating rich in seed cell-matrix, could induce local neo-vascularization, facilitating oriented SMC remodeling and lumen epithelialization as well as patency. Our findings indicate a central role of the synergistic interplay of seed cell-matrix bio-interface and nano-topographic cues in the vascularized urethral reconstruction.