Extracellular vesicles (EVs) are crucial mediators of intercellular communication and can be classified based on their physical properties, biomolecular structure, and origin. Among EVs, exosomes have garnered significant attention due to their potential as therapeutic and diagnostic tools. Exosomes are released via fusion of multivesicular bodies on plasma membranes and can be isolated from various biofluids using methods such as differential ultracentrifugation, immune affinity capture, ultrafiltration, and size exclusion chromatography. Herein, an overview of different techniques for exosome characterization and isolation, as well as the diverse applications of exosome detection, including their potential use in drug delivery and disease diagnosis, is provided. Additionally, we discuss the emerging field of exosome detection by sensors, which offers an up-and-coming avenue for point-of-care diagnostic tools development. Overall, this review aims to provide a exhaustive and up-to-date summary of the current state of exosome research.