<p>Afterglow luminescence imaging relies on the detection of photons from chemical or lattice defects after cessation of irradiation, enabling autofluorescence-free biomedical imaging with a higher signal-to-background ratio compared to fluorescence imaging. In particular, organic afterglow probes benefit from biocompatibility and can be designed with diverse molecular architectures and for various irradiation sources, including light, ultrasound and X-rays. In this Review, we first introduce the mechanisms governing afterglow emission. We then examine design strategies for organic afterglow probes, outlining strategies to improve their afterglow performance, particularly afterglow intensity, extended emission wavelengths, responsivity and diverse excitation sources, to allow bioimaging with high sensitivity and specificity in deep tissues. Finally, we highlight key biomedical applications in disease diagnosis and therapy and provide an overview of remaining challenges and opportunities of organic afterglow imaging.</p>