Two-dimensional (2D) cell culture is not ideal for traditional drug screening, because 2D culture does not accurately mimic the physiological microenvironment of tumor cells. Thus, a drug-screening system which more closely mimics the microenvironment of in vivo tumors is necessary. Here, we present a biomimicking bilayer microfluidic device that can facilitate antitumor drug screening. The microfluidic device consists of two polydimethylsiloxane (PDMS) pieces with channels which are separated by a semipermeable membrane to allow water, oxygen, and nutrition supply, while preventing cell migration. The channels embedded on the two PDMS pieces overlap each other over a long distance to ensure a larger exchange area to mimic the blood vessel-tumor model. High concentrations of endothelial cells (EC) are first seeded onto the membrane through the apical channel, and after a two-day culture, a confluent EC monolayer forms. Tumor spheroid-laden Matrigel is then seeded into the basal channel. After the Matrigel is cured, the device is ready for drug testing. Paclitaxel is used as the model drug for testing. Confocal microscopy and ImageJ are used to assess the efficacy of different concentrations of paclitaxel, and optical coherence tomography (OCT) is employed to determine the tumor volumetric change after the drug treatment. The results indicate that the proposed bilayer microfluidic device in combination with confocal and OCT optical characterization provide an efficient platform for antitumor drug testing.