A novel ratiometric fluorescent chemodosimeter has been developed for reusable detection of Hg2+. The chemodosimeter responds to Hg2+ sensitively and selectively with a remarkable fluorescence change from green to blue through hampering the excited state intramolecular proton transfer (ESIPT) process. This recyclable chemodosimeter can remove Hg2+ from water by forming a unique mercury-containing compound, which could be reused in the presence of NaBH4. Moreover, the chemodosimeter exhibits a ratiometric fluorescence response to Hg2+ with a very low detection limit (1.0 ppb), and it can be used to detect Hg2+ in drinking water. Furthermore, the ratiometric chemodosimeter has been successfully used for imaging Hg2+ in living cells and tissues using two-photon fluorescence microscopy due to the remarkable emission change from green to blue. This provides a novel testing method for detecting Hg2+ in living cells and tissues with low cytotoxicity and autofluorescence.