Perfusable vasculatures are essential for engineering three-dimensional thick tissues and organs in the fields of tissue engineering and regenerative medicine. Here, we describe an approach for the fabrication of double-layered vascular like structures (DVSs) composed of a monolayer of human vascular endothelial cells (HUVECs) covered with a dense human smooth muscle cell (SMC) layer. HUVECs were attached to a gold needle via the oligopeptide self-assembled monolayer and grown to form a HUVEC monolayer that was subsequently embedded in a photo-crosslinkable gelatin hydrogel containing SMC spheroids in a culture chamber. During four days of culture, the hydrogel significantly contracted and formed a dense SMC layer around the needle. The binding between the HUVEC layer and the gold needle was cleaved by applying a negative potential to desorb the oligopeptide and the needle was extracted from the chamber, resulting in a perfusable DVS composed of HUVEC and SMC layers. The DVS was cultured under perfusion, and the cells in the DVS showed greater expressions of SMC-specific genes compared to those of spheroids. The DVS possessed a dynamic contraction ability in response to acetylcholine as observed in the in vivo SMC layer. This study proposes a promising approach for the fabrication of perfusable vasculatures for the engineering of fully vascularized tissues and organs. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.