Inspired by the suitability of rhodamine spirolactam switching for superresolution imaging and fluorescent probe development, we herein synthesized rhodamine-based probes with sulfur-bearing amine and amino acid moieties and screened their abilities to respond to Hg2+ in the presence of other metal ions. The probe based on S-methylcysteine methyl ester–substituted rhodamine 6G (Rho6G-6) was identified as optimal because of its straightforward synthesis and rapid, selective, and sensitive (detection limit = 8 × 10–9 M) response to Hg2+ manifested by absorption spectrum and emission color changes. The complexation of Hg2+ by Rho6G-6 was examined by absorbance and fluorescence analyses, electrospray ionization–mass spectrometry, and 1H nuclear magnetic resonance (NMR) titration experiments. Furthermore, the practical applicability of the above probe was demonstrated by naked-eye colorimetric Hg2+ detection using paper test strips and the fluorescence-based qualitative mapping of the Hg2+ distribution in living HeLa cells and the organs of live mice. Thus, unlike other probe design strategies, the use of flexible S-methyl amino acids for Hg2+ binding provides new opportunities and strategies for photopromoting the ring switching of the rhodamine spirothiolactam.