The blood-brain barrier (BBB) controls chemical access to the brain and maintains fluid homeostasis, but in vitro models accurately simulating the physiological characteristics of the BBB are lacking. Here, we describe a simple and reproducible biomimetic chip-based model of the human BBB. In this bilayer co-culture, astrocytes and brain microvascular endothelial cells (BMECs) are respectively seeded in upper and lower chambers separated by a semi-permeable membrane, with fluid shear force provided by a precision tilt shaker. Evaluation of barrier crossing by fluorescein sodium, 40 kDa or 70 kDa FITC-dextran, or adeno-associated virus (AAV) particles demonstrates that this bilayer model provides similar or greater barrier function than Transwell assays. Comparison of AAV serotypes indicated that AAV-PHP.eB can cross the human BBB in vitro, and at higher efficiency than AAV9. Additionally, RNAi knockdown and virus capsid protein binding assays show that AAV-PHP.eB delivery is facilitated by receptor protein lymphocyte antigen-6E (LY6E) in humans. This in vitro model system uses a miniaturized chip to enable high-throughput investigations of AAV crossing efficiency in the BBB, and provides strong initial evidence that human LY6E mediates AAV-PHP.eB crossing the BBB.