AbstractCancer theranostics holds potential promise for precision medicine; however, most existing theranostic nanoagents are simply developed by doping both therapeutic agents and imaging agent into one particle entity, and thus have an “always‐on” pharmaceutical effect and imaging signals regardless of their in vivo location. Herein, the development of an organic afterglow protheranostic nanoassembly (APtN) that specifically activates both the pharmaceutical effect and diagnostic signals in response to a tumor‐associated chemical mediator (hydrogen peroxide, H2O2) is reported. APtN comprises an amphiphilic macromolecule and a near‐infrared (NIR) dye acting as the H2O2‐responsive afterglow prodrug and the afterglow initiator, respectively. Such a molecular architecture allows APtN to passively target tumors in living mice, specifically release the anticancer drug in the tumor, and spontaneously generate the uncaged afterglow substrate. Upon NIR light preirradiation, the afterglow initiator generates singlet oxygen to react and subsequently transform the uncaged afterglow substrate into an active self‐luminescent form. Thus, the intensity of generated afterglow luminescence is correlated with the drug release status, permitting real‐time in vivo monitoring of prodrug activation. This study proposes a background‐free design strategy toward activatable cancer theranostics.