Afterglow, an intrinsic phenomenon of persistent luminescence emitted from chemical defects after light irradiation, has shown tremendous promise for applications in bioimaging with an ultra-high signal-to-background ratio (SBR) in vivo. In contrast to inorganic phosphor materials, organic afterglow substrates possess high biocompatibility and structural diversity for the construction of molecular afterglow imaging probes with an ideal intensity, wavelength, and duration for in vivo imaging. In this tutorial review, we aim to introduce the recent advances in molecular afterglow imaging with a comprehensive summary of the reported afterglow substrates and mechanisms. Molecular designs of multicomponent afterglow imaging probes are also introduced with their biomedical applications in disease diagnosis and treatment. Lastly, future perspectives and potential challenges of molecular afterglow imaging in preclinical uses and clinical translations are discussed.