Exosomes are nano-sized vesicles released from cells into bodily fluids, carrying specific cargo of proteins, lipids, and genetic materials. They can be taken up by neighboring or distant cells, reprogramming recipient cells. The regulated formation of exosomes, their specific cargo, and cell-targeting specificity make them promising as non-invasive diagnostic biomarkers and therapeutic nanocarriers. This review discusses recent progress in understanding exosome biogenesis, molecular composition, and research technologies. Exosomes are generated from late endosomes, with the ESCRT pathway playing a crucial role in their formation. Tetraspanins and lipids also contribute to exosome biogenesis. Exosomes contain a complex mixture of proteins, lipids, and RNAs, which can influence recipient cells through various mechanisms. They have been implicated in immune regulation, wound healing, and disease progression. Exosome purification methods include differential centrifugation, size exclusion chromatography, and immunoaffinity capture. Exosomes have been used as diagnostic biomarkers in various diseases and as drug delivery vehicles. However, challenges remain in isolating, purifying, and quantifying exosomes, as well as standardizing their clinical application. Further research is needed to optimize these processes and enhance the therapeutic potential of exosomes.Exosomes are nano-sized vesicles released from cells into bodily fluids, carrying specific cargo of proteins, lipids, and genetic materials. They can be taken up by neighboring or distant cells, reprogramming recipient cells. The regulated formation of exosomes, their specific cargo, and cell-targeting specificity make them promising as non-invasive diagnostic biomarkers and therapeutic nanocarriers. This review discusses recent progress in understanding exosome biogenesis, molecular composition, and research technologies. Exosomes are generated from late endosomes, with the ESCRT pathway playing a crucial role in their formation. Tetraspanins and lipids also contribute to exosome biogenesis. Exosomes contain a complex mixture of proteins, lipids, and RNAs, which can influence recipient cells through various mechanisms. They have been implicated in immune regulation, wound healing, and disease progression. Exosome purification methods include differential centrifugation, size exclusion chromatography, and immunoaffinity capture. Exosomes have been used as diagnostic biomarkers in various diseases and as drug delivery vehicles. However, challenges remain in isolating, purifying, and quantifying exosomes, as well as standardizing their clinical application. Further research is needed to optimize these processes and enhance the therapeutic potential of exosomes.