We present an extracellular matrix(ECM)-based microfluidic device that creates a spatial chemical gradient by interstitial flow. Cells located on surfaces of ECM, such as vascular and skin tissues, are influenced by external factors, including interstitial flows, but few methods have been available to recapitulate such conditions. To address this gap, we developed a microfluidic device that can expose cells cultured on the surface of ECM to a continuous concentration gradient created by the interstitial flow. With a 3D printed water-soluble sacrificial mold, we readily fabricated ECM-based microfluidic devices embedding micromixers and a gradient generator. ECM mimicked the permeability of in vivo environments. Our device will serve as a platform to study cellular phenomena occurring on the surface of ECM, such as vascularization and migration of cancer cells.