The glymphatic system of brain waste clearance consists of the perivascular bulk convective fluid flow of cerebrospinal fluid (CSF) and interstitial fluid (ISF) and is responsible for fluid and solute clearance in the brain parenchyma, a portion of the human body without true lymphatic vessels. Key components of this system include aquaporin-4 (AQP4)-rich astrocyte endfeet and the basement membrane of cortical blood vessels creating a perivascular space (PVS) that allows CSF flow influx and ISF efflux. This system was only recently characterized in in vivo mouse models with later investigations with human patients via medical physics imaging modalities yet lacked a three-dimensional (3D) microfluidic in vitro model until this study. By creating a microfluidic biomimetic in vitro organ-on-chip model of the glymphatics system (glymphatics-on-chip) with primary human astrocytes and blood endothelial cells (BECs) housed a biomimetic extracellular matrix (ECM) with inducible ISF, we were able to investigate the regulation of dystrophin-associated complex (DAC) components with the presence of ISF, concluding that changes in AQP4 endfeet polarization may be ISF dependent via the DAC mechanism. Alzheimer’s disease (AD) is marked by the aggregation of extracellular amyloid-β (Aβ) and hyperphosphorylated tau (p-tau) as well as astrocyte dysfunction. For Aβ oligomers (oAβ) or aggregates to be formed, there must be Aβ monomers (mAβ) present; however, the roles of these isoforms in astrocyte pathogenesis are poorly understood. We cultured astrocytes in our 3D ECM and revealed that both isoforms caused astrocytic atrophy and hyper-reactivity but showed distinct Ca2+ changes in astrocytes. This was further explored with our glymphatics-on-chip model, which not only reproduced the astrocytic atrophy, hyper-reactivity, and Ca2+ changes induced by mAβ and oAβ, but recapitulated that the components of the DAC and AQP4 were dysregulated by mAβ and oAβ. Collectively, mAβ and oAβ cause distinct AD pathophysiological characteristics in the astrocytes. P-tau caused changes in astrocyte morphology in addition to vasoconstriction in the engineered blood vessels (BVs) and impaired glymphatic clearance, confirming findings from both in vivo models and patient samples. Altogether, our model provides a novel and innovative platform to further investigate the glymphatics system and AD with fewer animal studies.