Extracellular vesicles (EVs), isolated through techniques such as liquid biopsy, have emerged as crucial biomarkers in various diseases, including cancer. EVs were dismissed initially as cellular debris, EVs are now recognized for their role in intercellular communication, carrying proteins, RNAs, and other molecules between cells. Their stability in biofluids and ability to mirror their parent cells' molecular composition make them attractive candidates for non-invasive diagnostics. EVs, including microvesicles and exosomes, contribute to immune modulation and cancer progression, presenting both therapeutic challenges and opportunities. However, despite advances in analytical techniques like high-resolution microscopy and nanoparticle tracking analysis (NTA), standardization in EV isolation and characterization remains a hurdle. Cutting-edge technologies, such as atomic force microscopy and Raman tweezers microspectroscopy, have enhanced our understanding of single EVs, yet issues like low throughput and high technical complexity limit their widespread application. Other technologies like transmission electron microscopy, cryogenic transmission electron microscopy, super-resolution microscopy, direct stochastic optical reconstruction microscopy, single-molecule localization microscopy, tunable resistive pulse sensing, single-particle interferometric reflectance imaging sensor, flow cytometry, droplet digital analysis, total internal reflection fluorescence also contribute to EV analysis. Future research must focus on improving detection methods, developing novel analytical platforms, and integrating artificial intelligence to enhance the specificity of EV characterization. The future of EV research holds promise for breakthroughs in precision medicine, with a collaborative effort needed to translate these advancements into clinical practice.