Ligand-protected metal nanoclusters (NCs) having hydrodynamic diameters and fluorescent properties have reasonable potential for ion detection and bio-imaging applications by taking advantage of their smaller sizes and degradability as compared with larger nanoparticles. By controlling the pH value, here we have synthesized a fluorescent silver nanocluster, Ag–glutathione, which is found to be a versatile chemosensor operative for the detection of both manganese (Mn²⁺) and iodine (I⁻) ions. Mn²⁺ and I⁻ are further distinguished by the ratiometric absorption spectrometry method. Care was taken for sufficient spectral analyses, based upon which we have fully demonstrated the chemosensing mechanisms of Ag@glutathione (Ag@SG) NCs applied in ion detection. Having expounded this issue, we further investigated the potential application of Ag@SG NCs in bio-imaging. It is interesting to find that the as-prepared Ag@SG NCs are nontoxic and available for fluorescence imaging of MCF-7 cells, with on/off alternation of fluorescence emission in the presence of Mn²⁺ or I⁻ ions. The low cytotoxicity, good penetrability and on/off fluorescence property of Ag@SG NCs in MCF-7 cells suggest promising biological applications.