A Hg2+-selective fluorescent sensor, RosHg, has been developed based on a rosamine platform. RosHg exhibited a similar to 20-fold increase in fluorescence emission upon binding with Hg2+, and the enhanced fluorescence was immediately decreased when glutathione was added to a solution of the Hg-RosHg complex. The dissociation constant for the Hg2+ complex was determined to be 0.10 fM by using a set of Hg2+/Mg2+/ethylenediaminetetra-acetic acid buffer solutions. Confocal microscopy experiments demonstrated that this sensor can monitor changes of the Hg2+ level in the mitochondria of living cells.