Most studies of cancer metastasis focus on cancer cell invasion utilizing adhesion assays that are performed independently, and are thus limited in their ability to mimic complex cancer metastasis on a chip. Here we report the development of an integrated cell-based microfluidic chip for intra- and extravasation that combines two assays on one chip for the study of the complex cascade of cancer metastasis. This device consists of two parts; one is an intravasation chamber for the three-dimensional (3-D) culture of cancer cells using a Matrigel matrix, and the other is an extravasation chamber for the detection of metastasized cancer cells by adhesion molecules expressed by epithelial cells. In this novel system, the intravasation and extravasation processes of cancer metastasis can be studied simultaneously using four screw valves. Metastatic LOVO and non-metastatic SW480 cells were used in this study, and the invasion of LOVOs was found to be higher compared to SW480. In contrast, invasion of cells treated with metalloproteinase (MMP) inhibitors decreased within the intravasation chamber. Degraded cancer cells from the intravasation chamber were detected within the extravasation chamber under physiological conditions of shear stress, and differences in binding efficiency were also detected when CA19-9 antibody, an inhibitor of cancer cell adhesion, was used to treat degraded cancer cells. Our results support the potential usefulness of this new 3D cell-based microfluidic system as a drug screening tool to select targets for the development of new drugs and to verify their effectiveness.