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Copper is an essential element in living organisms and the monitoring of copper ions in aquatic environments and biological systems is of great significance. Inspired by a family of visible light responsive photoswitchable compounds, we report here a fluorescent probe (HTI-Q) for Cu2+ with excellent selectivity, high sensitivity, and a limit of detection (LOD) of ca. 0.02 μM. While the central carbon-carbon double bond of HTI-Q is photoisomerizable between the Z and E configurations, it is locked upon binding with Cu2+ into the E configuration, resulting in a large bathochromic shift (102 nm) and ratiometric fluorescence changes. Different sensing modes were demonstrated including in mixed solvents, a two-phase sensing system containing cation exchanger, and fluorescent nanoprobes containing a reference dye. The absorbance change of HTI-Q upon 470 nm light illumination also depended on the concentration of Cu2+. HTI-Q-based sensors were successfully applied to determine Cu2+ in real water samples with excellent recovery. Further, fluorescent nanoprobes incorporating HTI-Q were successfully applied to image endolysosomal Cu2+ changes upon external stimulation. [Display omitted] • A photoswitchable Cu2+ probe (HTI-Q) was proposed for the first time based on hemithioindigo. • HTI-Q offered tunable sensitivity, high selectivity, and several different optical sensing modes. • Nanoprobes containing HTI-Q were successfully applied for subcellular fluorescence imaging in the endolysosomes. [ABSTRACT FROM AUTHOR]