A rhodamine-B based chemosensor with diphenylselenium, RhoSe, has been synthesized, and its detection behavior towards various metal ions is studied via UV/Vis and fluorescence spectroscopy. RhoSe shows a selective response to Hg 2+ in CH 3 OH/H 2 O (v/v = 9:1) solutions over other metal ions. After addition of Hg 2+ , the solution of RhoSe displays an obvious color change from colorless to pink and a significant, 48-fold fluorescence enhancement. The color change and fluorescence enhancement are attributed to the ring-opening of the spirolactam in the rhodamine fluorophore, which is induced by Hg 2+ binding. The binding ratio of RhoSe-Hg 2+ was determined by a Job plot as a 1:1 ratio, and the effective pH range for Hg 2+ detection was 4.0-10. Importantly, the reversibility of the RhoSe-Hg 2+ complex was observed through the addition of Na 2 S. For practical applications, the strip method was utilized to detect Hg 2+ in water/methanol solution. In addition, confocal fluorescence microscopy experiments demonstrated that RhoSe is an effective fluorescent probe for Hg 2+ detection in vitro and in vivo.