A simple Cu2+-selective ratiometric fluorescence signaling probe based on the hydrolysis of dansylhydrazine was developed. Dansylhydrazine 1 showed prominent ratiometric fluorescence signaling behavior toward Cu2+ ions in a 50% aqueous (pH 7.4) acetonitrile solution. The ratiometric signaling behavior was due to the Cu2+-assisted hydrolysis of the yellow-colored fluorescent dansylhydrazine to strongly blue-fluorescent dansyl acid. The Cu2+-selective signaling of probe 1 was not affected by the presence of biologically important metal ions. The ratiometric signaling of Cu2+ by 1 was possible with a detection limit of 6.0 × 10−8 M (3.8 ppb). In addition, the signaling of Cu2+ in biological samples using synthetic urine was also tested.