Mimicking the cellular microenvironment is important for organoids and organ on-a-chip studies. One of the current issues is to introduce vessel-like structures into the culture system to improve the cellular and tissue functions, which deserves particular efforts in design and systematic consideration. Based on a standard device configuration, we fabricated a vessel-like component which can be easily integrated for cell co-culture. This component consists of an embedded monolayer of gelatin nanofibers on the top of an open channel. It can then be enclosed with an upper plastic plate with molded chamber, channels and standard Luer connectors. Human umbilical vein endothelial cells (HUVECs) were firstly introduced into the vessel-like channel and cultivated three-dimensionally with the help of a rotational device. Then, a flow was applied for cytoskeleton remolding, resulted in a dense and aligned HUVECs layer. Afterward, human glioblastoma cells (U87) were introduced in the upside of the fiber layer and a flow was also applied for the upper cell layer culture. Our results show adjunct formation of HUVEC and U87 cell layers on both sides of the monolayer of gelatin nanofibers, thereby providing a reliable support for a variety of co-culture assays.