A simple S–S (disulfide)-bridged dimeric Schiff base probe, L, has been designed, synthesized, and successfully characterized for the specific recognition of Al³⁺ and Fe²⁺ ions as fluorometric and colorimetric “turn-on” responses in a dimethylformamide (DMF)-H₂O solvent mixture, respectively. The probe L and each metal ion bind through a 1:1 complex stoichiometry, and the plausible sensing mechanism is proposed based on the inhibition of the photoinduced electron transfer process (PET). The reversible chemosensor L showed high sensitivity toward Al³⁺ and Fe²⁺ ions, which was analyzed by fluorescence and UV–vis spectroscopy techniques up to nanomolar detection limits, 38.26 × 10^–9 and 17.54 × 10^–9 M, respectively. These experimental details were advocated by density functional theory (DFT) calculations. The practical utility of the chemosensor L was further demonstrated in electrochemical sensing, in vitro antimicrobial activity, molecular logic gate function, and quantification of the trace amount of Al³⁺ and Fe²⁺ ions in real water samples.