ABSTRACT Organic afterglow materials hold great promise for applications in bioimaging, sensing, and information encryption, yet the construction of advanced nanostructures like vesicles with long‐lived luminescence remains a formidable challenge. We pioneer the construction of organic afterglow vesicles via polymerization‐induced self‐assembly (PISA), integrating a thermally activated delayed fluorescence (TADF)‐type organic afterglow emitter into block copolymer nanostructures. The resulting vesicles exhibit well‐defined hollow morphologies, uniform size distribution, and high solid content up to 20%. They display significant room‐temperature afterglow with an emission lifetime exceeding 200 ms and a photoluminescence quantum yield (PLQY) of 20.8%. The afterglow mechanism is attributed to efficient TADF with a moderate intersystem crossing rate, where the triplet excited states are excellently protected by the rigid glassy vesicle walls. Moreover, the vesicles show rapid, reversible, and repeatable oxygen‐responsive behavior, making them promising for reusable oxygen sensing. This work establishes a versatile and scalable strategy for designing functional organic afterglow nanostructures with potential applications in bioimaging, sensing, and environmental monitoring.