Exosomes are nanosized membrane vesicles released by the fusion of multivesicular bodies (MVBs) with the plasma membrane. Initially considered as cellular waste, exosomes are now recognized as important mediators of cell-to-cell communication and play roles in both physiological and pathological processes. Recent research has highlighted their potential in therapy and diagnosis. Exosome biogenesis involves the formation of intraluminal vesicles (ILVs) in MVBs, their transport to the plasma membrane, and their fusion with it. Key processes include autophagy and lysosomal exocytosis. The ESCRT machinery is crucial for exosome formation, with proteins like Hrs, TSG101, and ALIX playing significant roles. Other factors, such as tetraspanins and lipids, also contribute to exosome biogenesis and release. Exosome release is regulated by various cellular mechanisms, including Rab GTPases, SNARE proteins, and lipid signaling pathways. The release of exosomes can be influenced by cellular stress, autophagy, and the balance between degradation and secretion. Methodological challenges in exosome studies include the need for precise isolation and quantification techniques, as well as the potential for contamination with other vesicles. Understanding the molecular mechanisms behind exosome biogenesis and release is essential for their clinical application. Current research continues to explore the complex roles of exosomes in health and disease, with ongoing efforts to improve methods for their study and application.Exosomes are nanosized membrane vesicles released by the fusion of multivesicular bodies (MVBs) with the plasma membrane. Initially considered as cellular waste, exosomes are now recognized as important mediators of cell-to-cell communication and play roles in both physiological and pathological processes. Recent research has highlighted their potential in therapy and diagnosis. Exosome biogenesis involves the formation of intraluminal vesicles (ILVs) in MVBs, their transport to the plasma membrane, and their fusion with it. Key processes include autophagy and lysosomal exocytosis. The ESCRT machinery is crucial for exosome formation, with proteins like Hrs, TSG101, and ALIX playing significant roles. Other factors, such as tetraspanins and lipids, also contribute to exosome biogenesis and release. Exosome release is regulated by various cellular mechanisms, including Rab GTPases, SNARE proteins, and lipid signaling pathways. The release of exosomes can be influenced by cellular stress, autophagy, and the balance between degradation and secretion. Methodological challenges in exosome studies include the need for precise isolation and quantification techniques, as well as the potential for contamination with other vesicles. Understanding the molecular mechanisms behind exosome biogenesis and release is essential for their clinical application. Current research continues to explore the complex roles of exosomes in health and disease, with ongoing efforts to improve methods for their study and application.