Methylmercury (CH₃Hg(I)), produced by the action of aquatic bacteria on inorganic mercury, is the most hazardous among the mercury species. To date, no ratiometric fluorescent probes have been reported for the detection of both CH₃Hg(I) and Hg(II) in aquatic environments and in live cells. Herein, we designed a novel fluorescent probe incorporating a peptide containing a histidine residue with self-assembly properties specific to both mercury species and a fluorophore that exhibits red-shifted emissions upon aggregation. The probe effectively detected Hg(II) and CH₃Hg(I) in aqueous solutions (1% DMSO) through ratiometric fluorescence sensing with visible-light excitation (445 nm). The probe exhibited high selectivity for Hg(II) and CH₃Hg(I) among 19 metal ions, rapid response times (<4 s for CH₃Hg(I)), low detection limits (12.5 nM for Hg(II) and 248.6 nM for CH₃Hg(I)), reversible sensing, and a broad operational pH range. As a result, the probe was successfully employed for rapid and real-time sensing of CH₃Hg(I) and Hg(II) in both aquatic environments and live cells through distinct ratiometric fluorescent changes. A comprehensive binding mode study using dynamic light scattering, IR and CD spectroscopy, and NMR spectroscopy revealed that the chelation of mercury species by the peptide with the metal-binding site and the fluorophore triggers the self-assembly of the complex, enabling fast and sensitive ratiometric detection of mercury species. The combination of a self-assembling peptide with a metal-binding site and a responsive fluorophore provides a valuable fluorescent sensing platform for the detection and quantification of specific analytes, particularly in complex matrices.