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 Hg²⁺ in CH₃OH/H₂O (v/v = 9 : 1) solutions over other metal ions. After addition of Hg²⁺, 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 Hg²⁺ binding. The binding ratio of RhoSe–Hg²⁺ was determined by a Job plot as a 1 : 1 ratio, and the effective pH range for Hg²⁺ detection was 4.0–10. Importantly, the reversibility of the RhoSe–Hg²⁺ complex was observed through the addition of Na₂S. For practical applications, the strip method was utilized to detect Hg²⁺ in water/methanol solution. In addition, confocal fluorescence microscopy experiments demonstrated that RhoSe is an effective fluorescent probe for Hg²⁺ detection in vitro and in vivo.