The adhesion of cancer cells to the vascular endothelium during hematogenous metastasis is a crucial first step, involving the interaction of multiple adhesion molecules between cancer cells and endothelial cells. Here, a parallel-plate flow chamber combined with fluorescence microscopy was used to observe the adhesion behavior and subsequent calcium response of MDA-MB-231 cells on different functionalized substrates under flows, revealing the underlying force-regulation mechanism by analyzing and extracting relevant characteristic parameters. Our results demonstrated that fluid shear stress positively regulated the rolling velocity of cells by affecting the dissociation rate constant of CD44/E-selectin, and rapidly activated integrin α5β1 at the sub-second level, slowing down the rolling velocity of cells, but not enough to firm adhesion. Force triggered the calcium response of MDA-MB-231 cells on E-selectin. Furthermore, the activated integrin α5β1 binding with fibronectin enhanced and quickened cellular calcium response with higher activation ratio and peak intensity, and shorter delay time. This study can deepen the understanding of the hematogenous metastasis process of breast cancer cells, and provide reference for relevant clinical treatment strategies and drug development.