DT-diaphorase is a cytosolic flavoenzyme whose level is strongly elevated in a number of tumor types. Incorporating a DT-diaphorase's substrate in the structure of anticancer drugs may facilitate cancer detection and therapy. Herein, we developed a novel pro-prodrug nanosystem for cancer detection and therapy, which features enzyme-activated fluorescence emission and subsequent light-triggered drug release. The pro-prodrug molecule comprises an anticancer drug methotrexate (MTX), an enzyme (DT-diaphorase) responsive quinone propionic acid moiety and a light-activatable coumarinyl. In the absence of DT-diaphorase, the quinone propionic acid moiety quenches the fluorescence of coumarin via photoinduced electron transfer (PET) and blocks the photocleavage pathway. DT-diaphorase can annihilate the effect of PET and restore the fluorescence of coumarin. This fluorescence serves as the reporting signal for assessing the enzyme biomarker level and discriminates tumor cells from normal cells, and subsequently photocontrollable release of the active drug, MTX, can be activated via one- or two-photon irradiation. This pro-prodrug nanosystem shows strong cytotoxicity toward cancer cells and a negligible effect on normal cells. This strategy provides a new platform for constructing nanosystems for cancer detection and subsequent on-demand selective killing of cancer cells via both internal- and external-stimuli activation.