Abstract Organic fluorophore‐based afterglow luminescence is gaining prominence as a powerful tool for biomedical imaging due to its excellent biocompatibility and high signal‐to‐background ratio. In contrast to the tedious synthesis of organic afterglow fluorophores (AFs), a versatile strategy is introduced to significantly enhance afterglow luminescence by encapsulating conventional AFs within a bovine serum albumin (BSA) matrix. Specifically, self‐sustained (Ce6) and multi‐component (TCPP‐HBA) AFs, encapsulated within the BSA matrix, exhibit a remarkable ten‐fold increase in afterglow intensity compared with the conventional amphiphilic polymer matrix (F127). Furthermore, co‐encapsulation of Ce6 with near‐infrared dye (NCBS) in BSA amplifies ultrasound‐induced afterglow luminescence by twelve‐fold relative to F127. The mechanism studies reveal that the electron‐donating BSA matrix enhances singlet oxygen generation, stabilizes the formed high‐energy dioxetane intermediate, and increases the fluorophore quantum yield, collectively driving superior afterglow luminescence intensity. Leveraging this enhanced afterglow luminescence, both targeted tumor imaging and small metastatic tumor foci detection are achieved under its guidance. The developed BSA encapsulation strategy offers the potential to greatly improve afterglow imaging performance under clinically excitable sources (e.g., ultrasound, X‐ray), thereby broadening their biomedical applications.