Hydrogels have garnered significant attention in the biomedical field due to their high-water absorption capacity, flexibility, and biocompatibility.

Arterial stenosis or blockage is the leading cause of cardiovascular disease, and the common solution is to substitute the arteries by autologous veins or bypass the blood vessels physically. With the development of science and technology, arteries with diameter larger than 6 mm can be substituted by unbiodegradable polymers, such as polytetrafluoroethylene, clinically. Nevertheless, the construction of a small-diameter (less than 6 mm) artery with living cells has always been a thorny problem. In this study, a suit of combined mold was designed and forged for constructing small-diameter arterial vessels. Based on this combined mold, bioactive arterial vessels containing adipose-derived stem cells (ASCs) and different growth factors (GFs) were assembled together to mimic the inner and middle layers of the natural arteries. Before assembling, ASCs and GFs were loaded into a gelatin/alginate hydrogel. To enhance the mechanical property of the bilayer arterial vessels, polylactic–glycolic acid (PLGA) was applied on the surface of the bilayer vessels to form the outer third layer. The biocompatibility, morphology and mechanical property of the constructed triple-layer arterial vessels were characterized. The morphological results manifested that cells grow well in the gelatin/alginate hydrogels, and ASCs were differentiated into endothelial cells (ECs) and smooth muscle cells (SMCs), respectively. In addition, under the action of shear stress produced by the flow of the culture medium, cells in the hydrogels with high density were connected to each other, similar to the natural vascular endothelial tissues (i.e., endothelia). Especially, the mechanical property of the triple-layer arterial vessels can well meet the anti-stress requirements as human blood vessels. In a word, a small-diameter arterial vessel was successfully constructed through the combined mold and has a promising application prospect as a clinical small-diameter vessel graft.