Organic afterglow material is an optical material possessing afterglow properties, and can emit light for an extended period following excitation, thereby increase imaging duration and sensitivity. Organic afterglow materials are commonly utilized in biological analysis due to their flexible design and good biocompatibility. Owing to the light-excitation-free luminescence, the afterglow luminescence can circumvent the interference of tissue auto-fluorescence, and provide higher signal-to-background ratio and sensitivity superior to fluorescence. This review provides an overview of the recent advances in organic afterglow materials, which comprehensively summarizes the reported medical applications of these materials in bioanalysis and afterglow imaging. Finally, the article discusses the prospects and challenges of using organic afterglow materials in molecular construction and clinical imaging.