 Exosomes are small lipid bilayer-enclosed particles secreted from most cell types. They are a subset of extracellular vesicles and have gained increasing attention over the past decade due to their emerging role as intracellular messengers and potential in disease diagnosis. Exosomes contain proteins, lipids, and RNA that are specific to their cell origin and can deliver cargo to both nearby and distant cells. This makes them promising candidates for drug delivery and disease diagnosis. However, challenges such as inefficient separation methods, difficulties in characterization, and lack of specific biomarkers have slowed progress in this relatively new field of research. In this review, we summarized the current knowledge on exosome biogenesis, their roles in disease progression, and therapeutic applications and opportunities in bioengineering. Additionally, we discussed the established and emerging technologies in exosome isolation and characterization. Finally, we identified key challenges in exosome research and provide directions for future studies. This article was authored by Ciali, Alexander L. Corbett, Irfan Tadizadi, and others. We are article.tv, links in the description below.