Long-lasting afterglow luminescence imaging that detects photons slowly being released from chemical defects has emerged, eliminating the need for real-time photoexcitation and enabling autofluorescence-free in vivo imaging with high signal-to-background ratios (SBRs). Organic afterglow nano-systems are notable for their tunability and design versatility. However, challenges such as unsatisfactory afterglow intensity, short emission wavelengths, limited activatable strategies, and shallow tissue penetration depth hinder their widespread biomedical applications and clinical translation. Such contradiction between promising prospects and insufficient properties has spurred researchers' efforts to improve afterglow performance. In this review, we briefly outline the general composition and mechanisms of organic afterglow luminescence, with a focus on design strategies and an in-depth understanding of the structure-property relationship to advance afterglow luminescence imaging. Furthermore, pending issues and future perspectives are discussed.