Exosomes, a type of extracellular vesicle with a diameter of approximately 100 nm that is secreted by all cells, regulate the phenotype and function of recipient cells by carrying molecules such as proteins, nucleic acids, and lipids and are important mediators of intercellular communication. Exosomes are involved in various physiological and pathological processes such as immunomodulation, angiogenesis, tumorigenesis, metastasis, and chemoresistance. Due to their excellent properties, exosomes have shown their potential application in the clinical diagnosis and treatment of disease. The functions of exosomes depend on their biogenesis, uptake, and composition. Thus, a deeper understanding of these processes and regulatory mechanisms can help to find new targets for disease diagnosis and therapy. Therefore, this review summarizes and integrates the recent advances in the regulatory mechanisms of the entire biological process of exosomes, starting from the formation of early-sorting endosomes (ESCs) by plasma membrane invagination to the release of exosomes by fusion of multivesicular bodies (MVBs) with the plasma membrane, as well as the regulatory process of the interactions between exosomes and recipient cells. We also describe and discuss the regulatory mechanisms of exosome production in tumor cells and the potential of exosomes used in cancer diagnosis and therapy.