Abstract: Glutathione (GSH) was introduced into rhodamine 6G derivative (Rh6G2) to prepare a fluorescent probe Rh6G2-GSH, and Hg²⁺ content in water was determined by fluorescence spectroscopy and ultraviolet-visible spectrophotometry based on the changes in fluorescence intensity and absorbance occurred in the process of the fluorescent probe for recognition of Hg²⁺. Rh6G2 and GSH were added into a buffer solution (pH 7.0) composed of HEPES solution and methane with volume ratio of 1:1, and the fluorescent probe of Rh6G2-GSH was synthesized by self-assembly reaction. The fluorescent probe was mixed with the water sample, and the mixed solution was measured under the optimized instrument working conditions of the two methods. The results showed that the fluorescent probe could recognize Hg²⁺ in a wide acidity range (pH 7-12), with a less interference produced by common metal cations; linearity ranges of the standard curves made by the two methods were found in the ranges of 2.5×10^-5-2.5×10^-4 mol·L^-1 and 5.0×10^-5-2.5×10^-4 mol·L^-1, with detection limits of 1.39×10^-9 mol·L^-1 and 4.07×10^-7 mol·L^-1. The recovery test was made on the actual samples by standard addition method at three concentration levels, giving recoveries in the ranges of 95.1%-120% and 94.8%-100%, and RSDs of determined values (n=5) were found in the ranges of 0.46%-2.4% and 0.15%-3.4%. Quadrupole-time-of-flight mass spectrometer was used to investigate the fluorescence detection mechanism, and it turned out that after Rh6G2-GSH was irreversibly combined with Hg²⁺, it would undergo further hydrolysis to give an organic acid with a higher fluorescence intensity.