The oral uptake of probiotic microorganisms as food additives is one widely used strategy to sustain and improve the homeostasis of intestinal microbiota that protect the intestinal epithelia from attack by pathogenic bacteria. Once delivered to the ileum and colon, probiotics must adhere and form colonies on mucus that coats the surface of intestinal epithelial cells. Although an increasing amount of knowledge about the genetic and molecular level mechanisms of probiotics-mucus interactions has been accumulated, little is known about the physicochemical aspects of probiotics-mucus interactions under physiological shear in intestines. In this study, we established well-defined models of intestinal epithelial cell monolayers based on two major constituents of gut epithelia, enterocytes and goblet cells. First, the formation of a polarized cell monolayer sealed by tight junctions was monitored by transepithelial electrical resistance over time. The establishment of tight junctions and secretion of mucus proteins (mucin) was confirmed by immunofluorescence staining. In the next step, we measured the elasticity of cell monolayer surfaces by indentation using particle-assisted atomic force microscopy. The effective elastic modulus of goblet cell-like cells was 30 times smaller compared to that of enterocyte-like cells, which can be attributed to the secretion of a 3 μm thick mucin layer. As probiotics, we used Lactobacillus rhamnosus GG (LGG), which is one of the most widely used strains as food additives. To investigate the dynamic adhesion of LGG to the intestine model surface, we transferred the epithelial cell monolayer into a microfluidic chamber. A distinct difference in dynamic adhesion between two cell types was observed, which could be attributed to the difference in the mucin expression amount. Remarkably, we found that the dynamic LGG adhesion is enhanced by the increase in shear stress, showing a maximum binding efficiency at 0.3 Pa. Finally, we examined the persistence of LGG adhesion by a stepwise increase in the shear stress exerted on adherent LGG, demonstrating that LGG could withstand high shear stress even beyond that of physiological stress. The obtained results present a large potential to quantitatively understand the influence of engineered foods and probiotics on the homeostasis of microbiota on the surface of intestinal epithelia.