rhodamine-based optical sensor Rh–3S has been developed for selective detection of Hg2+ in aqueous solution as well as in living cells. UV–vis and fluorescence spectroscopic studies reveal that Rh–3S showsmarked sensitivity and selectivity to Hg2+ with a chelation-induced “off–on” response in acetonitrile (ACN)/MOPS buffer (10mM, pH 7.3, v/v, 1:1). The Rh–3S sensor binds Hg2+ in a 1:1 stoichiometrywith an apparent binding constant 3.71× 16 M (log K= 6.57) and displays a distinct change in color and fluorescence upon the alteration of free Hg2+ levels in solutionwith a reversible response and little interferencewith other biological relevant metal ions. Live cell confocal imaging studies demonstrate that the sensor is also capable of imaging the presence of Hg2+ ions as well as its dynamic changes in live cells.