Cells secrete extracellular vesicles (EVs) through various biogenesis pathways, resulting in distinct molecular compositions even when originating from the same cells. Analyzing individual EVs is challenging due to the need to overcome issues such as nanoscale size, heterogeneity of EVs, and measurement accuracy. Overcoming these challenges in EV research not only advances the field of biopsy but also enables progress in the biological research of EVs. In this study, we analyze quantifying membrane proteins using a novel methodology based on single-molecule fluorescence spectroscopy. We used TIRF based fluorescence imaging or Fluorescence Correlation Spectroscopy (FCS) to observe antibodies or aptamers binding to single EVs. Our results demonstrate the quantification of various membrane proteins, including CD63 and CD81. Consequently, we develop a membrane protein quantification assay for individual EVs using single-molecule and particle fluorescence imaging spectroscopy. Through this single molecule assay, we will elucidate the distribution of membrane proteins in EVs derived from various tumor cells, potentially enabling their use in future biopsies or quantification studies.