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A rhodamine spirolactam derivative ( 1 ) bearing a hydrophilic carboxylic acid group is developed as a fluorescent chemodosimeter for bivalent mercury ions (Hg 2+ ) in 100% aqueous solution. It exhibits a highly sensitive “turn-on” fluorescent response toward Hg 2+ with a 42-fold fluorescence intensity enhancement under 1 equiv. of Hg 2+ added. The chemodosimeter can be applied to the quantification of Hg 2+ with a linear range covering from 3.0 × 10 −7 to 1.0 × 10 −5 M and a detection limit of 9.7 × 10 −8 M. Most importantly, the fluorescence changes of the chemodosimeter are remarkably specific for Hg 2+ in the presence of other metal ions, which meet the selective requirements for practical application. Moreover, the experiment results show that the response behavior of 1 towards Hg 2+ is pH independent in neutral condition (pH 5.0–8.0) and the response is fast (response time less than 3 min). Furthermore, the ring-opening mechanism of the rhodamine spirolactam induced by Hg 2+ was supported by NMR, MS, and DFT theoretical calculations. In addition, the proposed chemodosimeter has been used to detect Hg 2+ in water samples and image Hg 2+ in living cells with satisfying results. [ABSTRACT FROM AUTHOR]