A coumarin-derived complex, Hg2L2 was reported as a highly sensitive and selective probe for the detection of mercapto biomolecules in aqueous solution. The addition of Cys to a 99% aqueous solution of Hg2L2 resulted in rapid and remarkable fluorescence OFF-ON (emission at 525 nm) due to the ligand-exchange reaction of Cys with L coordinated to Hg2+. The increased fluorescence can be completely quenched by Hg2+ and recovered again by the subsequent addition of Cys. Such a fluorescence OFF-ON circle can be repeated at least 10 times by the alterative addition of Cys and Hg2+ to the solution of Hg2L2, indicating that it can be used as a convertible and reversible probe for the detection of Cys. The interconversion of Hg2L2 and L via the decomplexation/complexation by the modulation of Cys/Hg2+ was definitely verified from their crystal structures. Other competitive amino acids without a thiol group cannot induce any fluorescence changes, implying that Hg2L2 can selectively determine mercapto biomolecules. Using confocal fluorescence imaging, L/Hg2L2 as a pair of reversible probes can be further applied to track and monitor the self-detoxification process of Hg2+ ions in SYSS cells.