Microfluidic technology is an important research tool for investigating angiogenesis in vitro. Here, we fabricated a polydimethylsiloxane (PDMS) microfluidic device with five cross-shaped chambers using a coverslip molding method. Then, the perforated PDMS microhole arrays prepared by soft lithography were assembled in the device as barriers; a single microhole had a diameter of 100 μm. After injecting type I collagen into the middle gel chamber, we added a culture medium containing a vascular endothelial growth factor (VEGF) into the middle chamber. It would generate a linear concentration gradient of VEGF across the gel region from the middle chamber to the four peripheral chambers. Human umbilical vein endothelial cells (HUVECs) were then seeded on the microhole barrier. With VEGF stimulation, cells migrated along the inner walls of the microholes, formed annularly distributed cell clusters at the gel-barrier interface, and then three-dimensionally (3D) sprouted into the collagen scaffold. After 4 days of culture, we quantitatively analyzed the sprouting morphogenesis. HUVECs cultured on the microhole barrier had longer sprouts than HUVECs cultured without the barrier (controls). Furthermore, the initial distribution of sprouts was more regular and more connections of tube-like structures were generated when the microhole barrier was used. This study introduces a novel microfluidic device containing both microtopographic structures and 3D collagen. HUVECs cultured with the microhole barrier could form well-interconnected tube-like structures and are thus an ideal in vitro angiogenesis model.