Constructing in vitro blood-brain barrier (BBB) model provides an innovative approach for studying the pathophysiology of the brain and screening drugs. Although commercial Transwell was the simplest and most widely utilized in vitro model, reasonably mimicking essential characteristics of human BBB and dynamic monitoring BBB function remain a challenge. Herein, inspired by the highly permeable extracellular matrix membrane in human BBB, a novel in vitro BBB model was established by combining functionalized anodic alumina oxide (AAO) membrane with nanochannel electrochemistry (ANE-BBB). Benefiting from the topographical nanostructures and modified cell-adhesive peptide on the AAO surface, a tight endothelial barrier was formed, which can be directly visualized by phase-contrast microscope, and the barrier function can be real time monitored by nanochannel electrochemistry. More importantly, according to the current signal induced by the diffusion of redox species through the nanochannels toward the underlying electrode surface, dynamic evaluation of BBB-crossing behavior and precise screening of brain-targeted nanodrugs can be achieved. The constructed ANE-BBB overcomes the shortcomings of invisible cell culture, low permeability, and inaccurate real-time monitoring of screening drugs in traditional Transwell and provides a reliable tool for the design of nanodrugs to the central nervous system.