Tumor hypoxia is correlated with increased resistance to chemotherapy and poor overall prognoses across a number of cancer types. We present here a cancer cell-selective and hypoxia-responsive probe (fol-BODIPY) designed on the basis of density functional theory (DFT)-optimized quantum chemical calculations. The fol-BODIPY probe was found to provide a rapid fluorescence “off −on”response to hypoxia relative to controls,which lack the folate or nitro-benzyl moieties.In vitro confocal microscopy and flow cytometry analyses, as well as in vivo near-infrared optical imaging of CT26 solid tumor-bearing mice,provided support for the contention that fol-BODIPY is more readily accepted by folate receptor-positive CT26 cancer cells and provides a superior fluorescence “off –on ”signal under hypoxic conditions than the controls. Based on the findings of this study, we propose that fol-BODIPY may serve as a tumor-targeting,hypoxia-activatable probe that allows for direct cancer monitoring both in vitro and in vivo.