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Amyloid beta oligomers (AßO) are pivotal in Alzheimer's Disease (AD), cleared by microglia cells, as immune cells in the brain. Microglia cells exposed to AßO are involved with migration, apoptosis, phagocytosis, and activated microglial receptors through AßO, impacting cellular mechanobiological characteristics such as microglial adhesion strength to the underlying substrate. Herein, a label-free microfluidic device was used to detect advancing AD conditions with increasing AßO concentrations on microglia BV2 cells by quantitatively comparing the cell-substrate adhesion. The microfluidic device, acting as an AD model, comprises a single channel, which functions as a cell adhesion assay. To assess cell-substrate adhesion under different AßO concentrations of 1 µM, 2.5 µM, and 5 µM, the number of the cells attached to the substrate was counted by real-time microscopy when the cells were under the flow shear stress of 3 Pa and 7.5 Pa corresponding to Reynolds number (Re) of 10 and 25, respectively. The data showed that quantifying the cell-substrate adhesion using the microfluidic device could successfully identify conditions of advancing AßO concentrations. Our findings indicated that the increased incubation time with AßO caused reduced cell-substrate adhesion strength. Additionally, increased AßO concentration was another factor that weakened microglial interaction with the substrate. The quantification of cell-substrate adhesion using 3 Pa compared to 7.5 Pa clearly demonstrated advancing AßO in AD. This study using the chip provides an AD model for a deeper understanding mechanobiological behaviors of microglia exposed to AßO corresponding to diagnosed AD conditions under an in vitro microenvironment.