<p>The vascular network is integral to the developmental and metabolic processes of various tissues and functions as a systemic circulatory system that also interconnects organs throughout the body. In this study, we describe a multilayered microfluidic organ-on-a-chip platform designed for reproducing various three-dimensional (3D) vascularized microtissue models for biological applications. This platform utilizes a porous membrane as a physical barrier and leverages capillary action for hydrogel self-filling. Its high flow resistance mitigates the risk of gel bursting into the medium channels and facilitates the delivery of substances to generate a wide range of interstitial flow and biochemical factor concentration gradients. This study demonstrated that this platform can be used to accurately replicate 3D microenvironments for vasculogenesis, angiogenesis, and vascularized tumor modeling. We also investigated the critical role of multiple microenvironmental regulations in vascular formation on a chip. Moreover, we reproduced the process of tumor angiogenesis, including primary solid tumor features and the inhibitory effects of antitumor drugs on tumor growth and tumor vasculature before and after angiogenesis. Hence, our multilayered microfluidic platform is valuable for exploring multiple vascular mechanisms and constructing specific microtissues that closely mimic in vivo physiological conditions, providing new strategies for cancer research. Furthermore, the multilayered configuration improves design flexibility and scalability, providing the potential for a multi-organ interconnected platform for high-throughput drug screening.</p>