Microfluidic technology is effective for constructing artificial microvascular tissues in vitro. A three-layer microfluidic chip was reported to use a polycarbonate (PC) porous membrane to separate culture fluid channels from tissue chambers. The micro-porous structure of the PC porous membrane formed a capillary bursting valve in the vertical direction to allow the hydrogel to fill the tissue chambers under the surface tension of the fluid and keep it from leaking into the culture fluid channels to ensure that the three-dimensional fluid microenvironment was effectively constructed. A COMSOL finite element model was established to simulate the three-dimensional fluidic microenvironment of a sin-gle rectangular chamber, and it was used to demonstrate that the three-layer microfluidic chip could provide a flow environment to stimulate blood vessel formation. The vascular differentiation abilities of endothelial progenitor cells and human umbilical vein endothelial cells were tested using the proposed three-layer microfluidic chip, and multiple shapes of microvessel networks cultured for up to approximately eight days were constructed on top of the multi-tissue chamber array chip. This three-layer microfluidic chip had the design flexibility to change the shape of the tissue chambers to provide different fluidic conditions at high throughput and provided a potential engineering tool to study the effects of fluidic factors on microvascular growth.