We present an extracellular matrix (ECM)-based stretchable microfluidic system for culturing in vitro three-dimensional (3D) vascular tissues, which mimics in vivo blood vessels. Human umbilical vein endothelial cells (HUVECs) can be cultured under perfusion and stretch simultaneously with real-time imaging by our proposed system. Our ECM (transglutaminase (TG) cross-linked gelatin)-based microchannel was fabricated by dissolving water-soluble sacrificial polyvinyl alcohol (PVA) molds printed with a 3D printer. Flows in the microchannel were analyzed under perfusion and stretch. We demonstrated simultaneous perfusion and stretch of TG gelatin-based microchannels culturing HUVECs. We suggest that our TG gelatin-based stretchable microfluidic system proves to be a useful tool for understanding the mechanisms of vascular tissue formation and mechanotransduction.