Ratiometric　afterglow　luminescence　has　emerged　as　a　transformative　approach　for　quantitative　bio-imaging　and　disease　monitoring,　addressing　fundamental　limitations　in　conventional　imaging　techniques.　This　review　examines　recent　advancements　in　ratiometric　afterglow　probes,　concentrating　on　their　design　principles,　energy　transfer　mechanisms,　and　applications　in　disease　detection.　The　integration　of　dual-emission　capabilities　with　prolonged　luminescence　enables　reliable　quantification　by　eliminating　background　interference　and　providing　internal　calibration.　Recent　advances　have　demonstrated　exceptional　performance　in　monitoring　cancer　immunotherapy,　arthritis　progression,　and　brain　tumor　development　through　the　detection　of　specific　biomarkers.　Despite　significant　progress,　challenges　remain　in　material　design,　signal　stability,　and　clinical　translation.　The　review　examines　current　engineering　solutions　and　emerging　opportunities,　particularly　in　the　integration　of　artificial　intelligence,　multiplexed　sensing,　and　the　development　of　point-of-care　diagnostics.　These　advances　establish　ratiometric　afterglow　luminescence　as　a　powerful　platform　for　next-generation　biomedical　imaging　and　disease　monitoring　systems.　©　2025　Elsevier　B.V.