A new fluorescent probe RY was synthesized for the detection of Au3+ ions based on a rhodamine B derivative. The fluorescent probe showed good selectivity and sensitivity to Au3+ ions. Obvious color and fluorescence changes could be observed with the naked eye while the fluorescent probe reacted with the Au3+ ions. The detection limit of the probe was determined to be 36 ppb by the fluorescence titration; the excellent linear relationship suggests that the probe is potentially useful for quantitative detection of Au3+ in vitro. We also demonstrated its bioimaging application in both living cells and mice; this was the first time that a fluorescent probe was successfully applied to imaging Au3+ in living animals.