Conventional fluorescence imaging is constrained by tissue autofluorescence, limited penetration depth, photobleaching, and the need for continuous excitation. Afterglow imaging addresses these limitations by producing persistent luminescence for extended periods following excitation, enabling background-free, high-sensitivity imaging with elevated signal-to-background ratios. This review summarizes the rational design, synthesis, processing, and standardized characterization of organic afterglow nanoparticles (ANPs) for biomedical imaging and therapy. Key afterglow mechanisms, including chemically initiated electron exchange luminescence (CIEEL), reactive oxygen species-mediated energy transfer, and trap-assisted recombination, are examined in relation to how molecular structure, polymer composition, and nanoparticle formulation influence emission wavelength, intensity, persistence, and degradability. Recent developments in small-molecule, conjugated-polymer, and polymerization-based afterglow systems are discussed, along with scalable fabrication methods such as nanoprecipitation and self-assembly. Standardized physicochemical and optical characterization metrics essential for reproducibility and clinical translation are outlined. Biomedical applications of ANPs in image-guided surgery, lymph node mapping, cardiovascular disease detection, photothermal and photodynamic therapy, and ultrasound- and X-ray–activated afterglow are highlighted.