A new pH-responsive fluorescent probe has been reported based on protonation-activable resonance charge transfer. In aqueous solution, probe PQ-Lyso exhibits ratiometric detection of pH changes with a large hypsochromic shift of 76 nm and remarkable changes in the fluorescence intensity ratio (<i>R</i> = <i>F</i><sub>494 nm</sub>/<i>F</i><sub>570 nm</sub>, <i>R</i>/<i>R</i><sub>0</sub> = 105). Furthermore, PQ-Lyso, which is localized to lysosomes in living cells, can calibrate lysosomal pH using fluorescence ratiometry