Extracellular vesicles (EVs) are membrane-bound vesicles secreted by all cell types and present in various biological fluids. They actively participate in intercellular communication and pathological processes by transporting proteins and nucleic acids between cells; and their molecular cargos may reflect the health status of the cell of origin thus they constitute an emerging target for liquid biopsy in cancer diagnosis. Identifying the molecular cargos of EVs is critical to revealing their biological functions and clinical values, which, however, remains challenging due to their small sizes, high heterogeneity and low quantities of biomolecules carried by each EV.Herein, we developed a series of methods to amplify the signals from specific EV cargos using DNA nanostructure, and applied these methods to investigate the expression levels of various protein and miRNA targets in individual EV. In Chapter 2, we developed a single-EV flow cytometry analysis approach to realize single EV counting and phenotyping in a conventional flow cytometer for the first time. This method employs target-initiated engineering of DNA nanostructures on each EV. By illuminating multiple markers on single EVs, statistically significant differences are revealed among the molecular signatures of EVs originating from several breast cancer cell lines, and the cancer cell-derived EVs among the heterogeneous EV populations are successfully recognized. In Chapter 3, we developed an ultrasensitive method to detect single EVs with an input as low as 100 vesicles/µL using fluorescence microscopy. Taking advantage of both DNA nanostructure labeling and EV membrane staining, this method can also permit calibration-free analysis of the protein profiles among different EV samples, leading to clear EV differentiation by their cell of origin. Moreover, this method allows simple co-localization of dual protein markers on the same EV, and the increased number of EVs carrying dual tumor proteins present in human serum could differentiate cancer patients from healthy controls at the early developmental stage. In Chapter 4, we developed a method to detect the miRNA cargos enclosed in individual EVs. This method employs nano-stir bars (NSB) for rapid EV capture and isolation from biological fluids. They also permit confinement of miRNA cargos from the same EV onto the same NSB. In this way, DNA nanostructures can be constructed upon recognition of specific miRNA targets for analysis of the miRNA cargos carried by each EV. Again, EV differentiation by their cell of origin can be simply achieved by evaluating the exosomal miRNAs expression profiles among different EV samples. Moreover, our method can provide real-time monitoring of EV secretion and the enclosed miRNA cargos in cell culture medium, facilitating analysis of EV biogenesis and functions. Overall, I developed various DNA nanostructure based methods for single EV analysis.