Optical imaging has the advantages of real-time monitoring, non-invasive, and high spatial-temporal resolution, and has been widely used in the biomedical field. Afterglow luminescence refers to the phenomenon that the material can continue to emit visible light after stopping illumination. Based on this, afterglow imaging can significantly reduce the interference of spontaneous fluorescence in biological tissues, and become an important means to improve the imaging quality, signal-to-noise ratio, and sensitivity. In this paper, the main categories of organic afterglow luminescent materials mediated by reactive oxygen species are systematically reviewed, and their luminescent mechanisms are discussed. The applications of these materials in biomedical detection, imaging, and tumor treatment are comprehensively introduced.