The extracellular matrix (ECM) provides physical and chemical support to the surrounding cells. During cell growth, ECM secretion and network formation influence cell morphology, cell adhesion, cell-to-cell interactions, and cell migration. Microfluidics-based cell culture systems are limited by the integration of structural ECM into the device. We report the development of a cell-derived ECM-incorporated microfluidic device that can provide structural characteristics and biochemical components of cell-derived ECM. Using an on-chip decellularization process, we constructed an ECM sheet, secreted and deposited from monolayer-cultured mouse embryonic fibroblasts (NIH/3T3), inside the microfluidic device. ECM components (including collagens, fibronectin, laminin, and elastin) and mesh-type fibronectin fibrous architecture were maintained on the surface of the porous membrane of the microfluidic device after decellularization. To verify the usability of the fibroblast-derived ECM sheet integrated microfluidic device in a cell culture platform, we tested the recellularization of human umbilical vein endothelial cells (HUVEC) and analyzed HUVEC–ECM and HUVEC–HUVEC interactions. On the ECM sheet, HUVECs exhibited morphologies and focal adhesion features that were markedly different from those of other groups. We then explored the effect of the ECM sheet on HUVEC mechanosensitivity. An increase in fluid shear stresses led to focal adhesion and the polymerization and reorganization of HUVEC adherens junctions, similar to natural junctional development, whereas the control group exhibited stimuli-insensitive behaviors. We conclude that the decellularized ECM sheet-incorporated microfluidic device provides an in vivo-like physical and biochemical ECM microenvironment for microfluidics-based cell culture.