Exosomes, a subset of extracellular vesicles (EVs) with diameters ranging from 30 to 150 nanometers, were once considered mere cellular debris. However, over the past two decades, they have been recognized as fundamental mediators of intercellular communication. These lipid-bilayer-enclosed vesicles are generated through the endosomal pathway and carry a complex cargo of proteins, lipids, and nucleic acids (including DNA, mRNA, and non-coding RNAs) that reflects the physiological state of their cell of origin. Upon release into the extracellular space and subsequent uptake by recipient cells, exosomes can modulate a wide array of biological processes, including immune responses, neuronal communication, and tissue repair. This review comprehensively explores the biogenesis, cargo sorting, and release mechanisms of exosomes. We then delve into their multifaceted roles in both maintaining homeostasis and driving disease progression, with a particular focus on cancer (pro-tumorigenic signaling, metastasis, and drug resistance), neurodegenerative disorders (propagation of pathological proteins like α-synuclein and tau), and cardiovascular diseases. Furthermore, we discuss the dual nature of exosomes as both biomarkers for disease diagnosis and prognosis and as innovative therapeutic vehicles for targeted drug and nucleic acid delivery. Finally, we address the current challenges and future perspectives in the rapidly evolving field of exosome research, highlighting their immense potential in precision medicine.