The blood-brain barrier (BBB) is essential for maintaining central nervous system (CNS) stability, and neuroinflammation may cause the dysfunction of the BBB. MicroRNA-146a (miR-146a) is closely associated with neuroinflammation, which showed significant upregulation in response to lipopolysaccharide (LPS) induction. Elucidating the relationship between LPS-induced miR-146a expression and the BBB could decipher the mechanism of many neurological diseases. Here, we constructed an in vitro microfluidic human-BBB (μF-hBBB) chip consisting of human umbilical vein vascular endothelial cells (HUVECs) and human astrocyte (HAs) cells. A tetrahedral DNA framework (TDF-3MB) nanoprobe was used to label miR-146a in HUVECs on μF-hBBB chips before and after LPS induction, and the study revealed a significant increase in miR-146a expression after LPS induction. We believe that such a μF-hBBB chip is a promising in vitro platform for further use in understanding CNS diseases.