In this work, a novel strategy was developed to fabricate prevascularized cell-layer blood vessels in thick tissues and small-diameter blood vessel substitutes using three-dimensional (3D) bioprinting technology. These thick vascularized tissues were comprised of cells, a decellularized extracellular matrix (dECM), and a vasculature of multilevel sizes and multibranch architectures. Pluronic F127 (PF 127) was used as a sacrificial material for the formation of the vasculature through a multi-nozzle 3D bioprinting system. After printing, Pluronic F127 was removed to obtain multilevel hollow channels for the attachment of human umbilical vein endothelial cells (HUVECs). To reconstruct functional small-diameter blood vessel substitutes, a supporting scaffold (SE1700) with a double-layer circular structure was first bioprinted. Human aortic vascular smooth muscle cells (HA-VSMCs), HUVECs, and human dermal fibroblasts–neonatal (HDF-n) were separately used to form the media, intima, and adventitia through perfusion into the corresponding location of the supporting scaffold. In particular, the dECM was used as the matrix of the small-diameter blood vessel substitutes. After culture in vitro for 48 h, fluorescent images revealed that cells maintained their viability and that the samples maintained structural integrity. In addition, we analyzed the mechanical properties of the printed scaffold and found that its elastic modulus approximated that of the natural aorta. These findings demonstrate the feasibility of fabricating different kinds of vessels to imitate the structure and function of the human vascular system using 3D bioprinting technology.