The blood-brain barrier (BBB) restricts the uptake of many neuro-therapeutic molecules, presenting a formidable hurdle to drug development in brain diseases. We proposed a new and dynamic in vivo-like three-dimensional microfluidic system that replicates the key structural, functional and mechanical properties of the blood-brain barrier in vivo. Multiple factors in this system work synergistically to accentuate BBB-specific attributes-permitting the analysis of complex organ-level responses in both normal and pathological microenvironments in brain tumors. The complex BBB microenvironment is reproduced in this system via physical cell-cell interaction, vascular mechanical cues and cell migration. This model possesses the unique capability to examine brain metastasis of human lung, breast and melanoma cells and their therapeutic responses to chemotherapy. The results suggest that the interactions between cancer cells and astrocytes in BBB microenvironment might affect the ability of malignant brain tumors to traverse between brain and vascular compartments. Furthermore, quantification of spatially resolved barrier functions exists within a single assay, providing a versatile and valuable platform for pharmaceutical development, drug testing and neuroscientific research.