Exosomes distributed by extracellular vesicles carry various information highly consistent with cells, becoming a new type of biomarker for tumor screening. However, although conventional characterization technologies can quantify size and morphology for exosomes, they are limited in related fields such as function tracing, protein quantification at unit point, and microstructural information. In this paper, firstly, different exosome characterization methods are systematically reviewed, such as dynamic light scattering, nanoparticle tracking analysis, flow cytometry, electron microscope, and emerging super-resolution imaging technologies. Then, advances in applications are described one by one. Last but not least, we compare the features of different technologies for exosomes and propose that super-resolution imaging technology can not only take into account the advantages of conventional characterization techniques but also provide accurate, real-time, and super-resolution quantitative analysis for exosomes. It provides a fine guide for exosome-related biomedical research, as well as application in liquid biopsy and analysis techniques.