The integration of three-dimensional micropatterning with microfluidics provides a unique opportunity to create perfusable tissue constructs in vitro. Herein, we have used this approach to create a tumor-on-a-chip with an endothelial barrier. Specifically, we photopatterned a mixture of endothelial cells and cancer spheroids within a gelatin methacrylate (GelMA) hydrogel inside a microfluidic device. The differential motility of endothelial and cancer cells in response to a controlled morphogen gradient across the cell-laden network drove the migration of endothelial cells to the periphery while maintaining the cancer cells within the interior of the hydrogel. The resultant endothelial cell layer forming cell-cell contact via VE-cadherin junctions was found to encompass the entire GelMA hydrogel structure. Furthermore, we have also examined the potential of such a tumor-on-a-chip system as a drug screening platform using doxorubicin, a model cancer drug.