Afterglow imaging holds great potential for ultrasensitive biomedical imaging. As it detects photons after the cessation of real-time light excitation, autofluorescence can therefore be effectively eliminated. However, afterglow imaging is still in its infant stage due to the lack of afterglow agents with satisfactory lifetime, biocompatibility, and high luminescence brightness, particularly afterglow in the near-infrared region for in vivo applications. To address these issues, this study for the first time reports chlorin nanoparticles (Ch-NPs) emitting afterglow luminescence peaking at 680 nm with a half-life of up to 1.5 h, which is almost 1 order of magnitude longer than those of other reported organic afterglow probes. In-depth experimental and theoretical studies revealed that the brightness of the afterglow luminescence is strongly correlated with the singlet oxygen ( 1 O 2 ) capacity and the oxidizability of the chlorins. Benefitting from the ultralong half-life and the minimized imaging background, small metastatic tumor foci of 3 mm 3 were successfully resected under the guidance of the afterglow luminescence generated upon a single shot of activation prior to the injection, which was impossible for conventional nearinfrared fluorescence imaging due to tissue autofluorescence.