Herein, a collagen gel-coated and aligned nanofiber membrane named Col-ANM is developed, which remarkably improves endothelial barrier function by providing biochemical and topographical cues simultaneously. Col-ANM is fabricated by collagen gel coating process on an aligned polycaprolactone (PCL) nanofiber membrane, which is obtained by a simple electrospinning process adopting a parallel electrode collector. Human umbilical vein endothelial cells (HUVECs) cultured on Col-ANM exhibit remarkably enhanced endothelial barrier function with high expression levels of intercellular junction proteins of ZO-1 and VE-cadherin, a high TEER, and a cellular permeability compared with the artificial porous membranes in commercial cell culture well inserts. The enhanced endothelial barrier function is conjectured to be attributed to the synergistic effects of topographical and biochemical cues provided by the aligned PCL nanofibers and collagen gel in the Col-ANM, respectively. Finally, the reactive oxygen species is applied to the HUVEC monolayer formed on the Col-ANM to destroy the tight junctions between HUVECs. The destruction of the tight junctions is demonstrated by the decreased TEER value over time. Results indicate the potential of Col-ANM in modeling endothelial barrier dysfunction-related diseases.