Societal concerns over toxic mercury accumulation in humans from fish and other dietary and environmental sources provide motivation to develop new tools and tactics for mercury detection in a wide range of laboratory and field settings. Here we report the synthesis, properties, and application of a selective and sensitive small-molecule chemosensor for fluorescence screening of mercury levels in fish. Mercuryfluor-1 (MF1) is a water-soluble, fluorescein-based reagent that features excellent selectivity for Hg2+ over competing analytes and the largest turn-on fluorescence response to date (>170-fold increase) for reporting this heavy metal ion in aqueous solution. Combining this chemoselective Hg2+ probe with a microwave digestion protocol provides a facile method for assaying mercury levels in fish samples with mercury concentrations spanning 0.1 to 8 ppm, a range well matched with the United States Environmental Protection Agency (U.S. EPA) standard for the maximum safe level of mercury in edible fish (0.55 ppm).