<p>Achieving accurate detection of dual metal ions remains crucial challenging in the domains of ion sensing and cell imaging. Herein, glutathione (GSH)-modified MoS₂ quantum dots (QDs) were synthesized via a simple one-step hydrothermal method. Upon excitation at 326&#xa0;nm, the GSH-MoS₂ QDs emit blue fluorescence and exhibit significant fluorescence quenching in the presence of Fe³⁺ and Ag⁺ ions, with a linear response at low concentrations. The detection limits for Fe³⁺ and Ag⁺ were determined to be 105 nM and 67.7nM, respectively. Fluorescence lifetime measurements reveal a decrease upon addition of Fe³⁺ and Ag⁺ decreasing from 6.37 ns to 6.25 ns and 5.91 ns respectively, which suggests that the fluorescence quenching mechanism of GSH-MoS₂ QDs is likely due to a combination of both static and Förster Resonance Energy Transfer (FRET) processes for Fe³⁺ and Ag⁺. The absolute fluorescence quantum yield of GSH-MoS₂ QDs was determined to be 11.13%. Furthermore, GSH-MoS₂ QDs exhibit low cytotoxicity and good biocompatibility, and are successfully applied for the detection of Fe³⁺ and Ag⁺ ions in living cells, highlighting that GSH-MoS₂ QDs have potential for use as fluorescent probes in biological sensing and cell imaging applications.</p> Graphical Abstract <p></p>