Construction of in vitro vascular models is of great significance to various biomedical research, such as pharmacokinetics and hemodynamics, thus is an important direction in tissue engineering. In this work, a standing surface acoustic wave field was constructed to spatially arrange suspended endothelial cells into a designated patterning. The cell patterning was maintained after the acoustic field was withdrawn by the solidified hydrogel. Then, interstitial flow was provided to activate vessel tube formation. Thus, a functional vessel-on-a-chip was engineered with specific vessel geometry. Vascular function, including perfusability and vascular barrier function, was characterized by beads loading and dextran diffusion, respectively. A computational atomistic simulation model was proposed to illustrate how solutes cross vascular lipid bilayer. The reported acoustofluidic methodology is capable of facile and reproducible fabrication of functional vessel network with specific geometry. It is promising to facilitate the development of both fundamental research and regenerative therapy.