A novel rhodamine-based fluorescent probe L2 with two “S” groups was synthesized and characterized. As expected, L2 exhibited high selectivity and sensitivity for Hg2+ over other commonly coexistent metal ions. Its selectivity is excellent, and the detection limit was measured to be 1ppm. The significant changes in the fluorescence color could be used for naked-eye detection. Furthermore, fluorescence imaging experiments of Hg2+ ions in living EC-109 cells demonstrated its value of practical applications in biological systems.