As a multifunctional fluorescent nanomaterial, carbon dots (CDs) not only have small size, stable chemical properties, excellent photoluminescence characteristics, but also exhibit good biocompatibility and low toxicity. It has attracted considerable attention in the field of nanotechnology and biological science. CDs contain abundant functional groups on the surface, which not only retain part of the properties of raw materials, but also may have new photoelectric, catalytic, biomedical, and other functions. In this review, we systematically summarize the synthesis methods, modifications, optical properties, and main biological functions of CDs in recent years. The application of functionalized modified CDs in biological detection, biological imaging, photodynamic therapy, photothermal therapy, targeted therapy, drug delivery, gene delivery, protein delivery, and other biomedical fields is introduced. The latest progress of CDs with its own biomedical function in antioxidant, anti‐pathogen, and disease treatment is summarized. Finally, we discuss some problems in the practical application of CDs and look forward to the future development trend of self‐functional CDs combined with surface modification to achieve multimodal treatment of diseases.