Exosomes are small nanosized biovesicles that form when multivesicular bodies and the plasma membrane fuse and are released into the surrounding body fluids. They are best known for their multifunction in mediating intercellular communication by transferring various biomolecules, including DNA, RNAs, proteins, and lipids, in a short- and long-distance manner and have been identified as health and disease messengers. Importantly, exosomes are necessary for various physiological processes in health and disease. The generation of exosomes depends on the status of the disease, which usually exhibits opposite roles by inducing enhanced cellular stress and damage. Recently, exosome-based nanotechnologies have provided unprecedented opportunities to boost the developments of exosome-related biology, chemistry, pathology, and therapeutics in different diseases based on their unique structural/compositional/morphological characteristics for next-generation nanomedicines. Herein, we provide a comprehensive overview of the recent advances in exosome nanotechnology research, including their classification, isolation and preparation, constitution, biological function, and nanobiomedical applications in disease treatment and diagnosis. Furthermore, future prospects were also concluded. This review will provide more inspiration for promoting the development of exosome-based advanced theranostic nanoplatforms and nanotechnology.