To accelerate the fabrication of thick-walled autologous tissue-engineered vascular grafts (TEVGs), a novel biotube inserting technique was developed. After 2 weeks of subcutaneous embedding in rabbits, silicone rods (diameter, 3 mm; length, 35 mm) became encapsulated in connective tissues. Single-layered biotubes were obtained after removing the silicone rods. One silicone rod encapsulated in tissues was inserted into a single-layered biotube to form two layers of autologous tubular tissues. Three layers of autologous tubular tissues were also obtained by applying the same technique. Following a 2-week re-embedding procedure, two layers or three layers of autologous tubular tissues were integrated to form two-layered or three-layered TEVGs. Both wall thickness and burst pressure of three-layered TEVGs were significantly higher than those of two-layered and single-layered TEVGs (P < 0.05). The two-layered TEVGs could be applied as small-caliber vascular grafts, and three-layered TEVGs could be applied as medium- or large-caliber vascular grafts.