Extracellular vesicles (EVs) represent an important mode of intercellular communication in both disease and developmental biology, exposing their potential in diagnostics and therapeutics. Recently, aptamer-based sensors, i.e. aptasensors, have been gradually applied in EV analysis due to their high selectivity and sensitivity. A fluorescent aptasensor enables easy readout by flow cytometry (FCM) and has more accuracy and convenience than conventional immunoassays for EV analysis. Here, we develop a fluorescent aptasensor-based method for quantitative analysis of nano-sized membrane vesicles by using high-resolution FCM. EVs as small as 100 nm are detected and quantified using a dual-staining procedure with the fluorescent aptasensor targeting CD63 and a cytoplasmic dye. Nano-sized EVs derived from bone marrow mesenchymal stem cells, human neural stem cells and human cornea epithelial cells are analyzed, and the result shows that their amount varies from 6.79 × 106 mL-1 to 2.08 × 108 mL-1 in culture media. The technique is also used to evaluate the bioactivity of EVs and, in the future, it may develop into a versatile tool to analyze and quantify EVs from a variety of biological objects with conventional cytometric instruments.