Extracellular vesicles (EVs) are released from cells and can be taken up by other cells to mediate communication among distant cells. The process of vesicle uptake is initiated by the docking of the vesicles onto membrane proteins on cells, but a generalizable technique for quantitatively observing these vesicle–protein interactions (VPIs) is lacking. Here, we develop a technique that measures VPIs between single vesicles and cell-surface proteins using total internal reflection fluorescence microscopy. We first describe a simple procedure that can effectively label vesicles without complex purification. Subsequently, we quantify the interaction between the labeled vesicles and target proteins either attached to a surface or embedded in a lipid bilayer. By employing cell-derived vesicles (CDVs) and intercellular adhesion molecule-1 (ICAM-1) as a model system, we determine the binding affinity of vesicles toward the ICAM-1 depending on cell types of vesicle origin. Moreover, controlling the surface density of proteins also reveals robust support from a tetraspanin protein CD9, with a critical dependence on molecular proximity. We expect that VPI imaging will be a useful tool to dissect the molecular mechanisms of vesicle uptake and to guide the development of therapeutic vesicles.