Extracellular vesicles (EVs) play a key role in cell-cell communication and thus have great potential to be utilized as therapeutic agents and diagnostic tools. In this study, we implemented single-molecule microscopy techniques as a toolbox for a comprehensive characterization as well as measurement of the cellular uptake of HEK293T cell-derived EVs (eGFP-labeled) in HeLa cells. A combination of fluorescence and atomic force microscopy revealed a fraction of 68% fluorescently labeled EVs with an average size of ∼45 nm. Two-color single-molecule fluorescence microscopy analysis elucidated the 3D dynamics of EVs entering HeLa cells. 3D colocalization analysis of two-color direct stochastic optical reconstruction microscopy (dSTORM) images revealed that 25% of EVs that experienced uptake colocalized with transferrin, which has been linked to early recycling of endosomes and clathrin-mediated endocytosis. The localization analysis was combined with stepwise photobleaching, providing a comparison of protein aggregation outside and inside the cells.