As many nanoparticles (NPs) have been exploited as drug carriers to overcome the resistance of the blood-brain barrier (BBB), reliable in vitro BBB models are urgently needed to help researchers to comprehensively understand drug nanocarrier-BBB interaction during penetration, which can prompt pre-clinical nanodrug exploitation. Herein, we developed a microfluidic microphysiological model, allowing the analysis of BBB homeostasis and NP penetration. We found that the BBB penetrability of gold nanoparticles (AuNPs) was size- and modification-dependent, which might be caused by a distinct transendocytosis pathway. Notably, transferrin-modified 13 nm AuNPs held the strongest BBB penetrability and induced the slightest BBB dysfunction, while bare 80 nm and 120 nm AuNPs showed opposite results. Moreover, further analysis of the protein corona showed that PEGylation reduced the protein absorption, and some proteins facilitated the BBB penetration of NPs. The developed microphysiological model provides a powerful tool for understanding the drug nanocarrier-BBB interaction, which is vital for exploiting high-efficiency and biocompatible nanodrugs.