AbstractAfterglow materials have attracted much attention owing to their long luminescence lifetimes, large Stokes shifts, and emission without real‐time excitation. Compared with traditional organic afterglow materials, carbon dots (CDs), as a new afterglow material, have superior properties such as easy preparation, low toxicity, and low cost. The emission color of afterglow CDs can be regulated by external factors such as excitation wavelength, temperature, and time, which is highly significant for expanding the diversified applications to make them available for biotechnology and information applications. This review summarizes the research progress of multicolor afterglow CDs in recent years, including luminescence regulation strategies, preparation methods, and applications. First, the multicolor afterglow CDs are classified into three strategies: multicolor room temperature phosphorescence (RTP), thermally activated delayed fluorescence converted to RTP, and delayed fluorescence based on Förster resonance energy transfer, and the strategies for regulating their luminescence properties are analyzed. Second, the preparation methods of achieving multicolor afterglow CDs are summarized in both matrix‐free and matrix‐confined aspects. Then, applications in anticounterfeiting and information encryption, sensing and bioimaging are introduced in detail. Finally, the future challenges and opportunities of multicolor afterglow CDs are prospected to provide ideas for their controlled design and wide application.