Measuring ligand interactions with membrane proteins in single live cells is critical for understanding many cellular processes and screening drugs. However, developing such a capability has been a difficult challenge. Here, we employ evanescent scattering microscopy (ESM) to show that ligand binding to membrane proteins can change the cell adhesion properties, which are intrinsic cell properties and independent of random cell micromotions and ligand mass, thus allowing the kinetics analyses of both proteins and small molecules binding to membrane proteins in both single fixed and live cells. In addition, utilizing the high spatiotemporal resolution of ESM, the positions of cell adhesion sites can be tracked in real-time to analyze the cell deformations and migrations, thus providing a potential approach for understanding the cell activity during the ligand binding process in detail. The presented method may pave the road for developing a versatile and easy-to-use label-free detection strategy for in situ analysis of molecular interaction dynamics in living biosystems with single-cell resolution.