Monitoring mitochondrial derived copper(II) in live cells is highly demanded, but accurately detecting is unmet due to the interference with cytoplasmic copper(II). Herein, we have reported the design, synthesis and characterization of photocontrollable fluorogenic probe, MCu-3, which is equipped with a photo-labile group (nitrobenzyl group) and mitochondria targeting unit (triphenylphosphonium salt). This novel probe showed an intense fluorescence enhancement in response to copper(II) without interference from other metal cations in the biological condition (pH 6–9). The detection limit is 1.7 × 10−7 mol/L in HEPES buffer. The confocal fluorescence imaging results demonstrated MCu-3 can visualize mitochondrial copper(II) in live mammalian cells. The clear advantage of our photocontrollable method is successful to avoid the influence of cytoplasmic copper(II) during mitochondria specific detection.