We present the synthesis, properties, and biological applications of Ratio-Coppersensor-1 (RCS1), a new water-soluble fluorescent sensor for ratiometric imaging of copper in living cells. RCS1 combines an asymmetric BODIPY reporter and thioether-based ligand receptor to provide high selectivity and sensitivity for Cu+ over other biologically relevant metal ions, including Cu2+ and Zn2+ a ca. 20-fold fluorescence ratio change upon Cu+ binding, and visible excitation and emission profiles compatible with standard fluorescence microscopy Filter sets. Live-cell confocal microscopy experiments show that RCS1 is membrane-permeable and can sense changes in the levels of labile Cu+ pools within living cells by ratiometric imaging, including expansion of endogenous stores of exchangeable intracellular Cu+ triggered by ascorbate stimulation in kidney and brain cells.