There remains a lack of small-caliber tissue-engineered blood vessels (TEBVs) with wide clinical use. Biotubes were developed by electrospinning and in-body tissue architecture (iBTA) technology to prepare small-caliber TEBVs with promising applications. Different ratios of hybrid fibers of poly(l-lactic-co-epsilon-caprolactone) (PLCL) and polyurethane (PU) were obtained by electrospinning, and the electrospun tubes were then implanted subcutaneously in the abdominal area of a rabbit (as an in vivo bioreactor). The biotubes were harvested after 4 weeks. They were then decellularized and cross-linked with heparin. PLCL/PU electrospun vascular tubes, decellularized biotubes (D-biotubes), and heparinized combined decellularized biotubes (H + D-biotubes) underwent carotid artery allograft transplantation in a rabbit model. Vascular ultrasound follow-up and histological observation revealed that the biotubes developed based on electrospinning and iBTA technology, after decellularization and heparinization cross-linking, showed a better patency rate, adequate mechanical properties, and remodeling ability in the rabbit model. IBTA technology caused a higher patency, and the heparinization cross-linking process gave the biotubes stronger mechanical properties.