<p>A quinoline-thiourea conjugate probe, AQS, was developed to selectively detect Hg²⁺ and CN⁻ ions in a DMSO:H₂O (1:1) buffer solution using fluorescence spectroscopy. Probe AQS, upon binding with Hg²⁺, the photon-induced electron transfer (PET) process is inhibited, assisted by the restriction of the C-N bond rotation resulting in significantly enhanced fluorescence intensity. Conversely, CN⁻ binding to the two NH groups triggers electron transfer from hydrogen to CN⁻, modulating the PET process and enabling the quenching of fluorescence of the probe AQS. The binding stoichiometry of probe AQS with Hg²⁺ and CN⁻ was determined to be 1:1 and 1:2, respectively, using Job's plot method. Density functional theory (DFT) calculations yielded energy gaps of 3.792&#xa0;eV for probe AQS, and 0.758 and 0.711&#xa0;eV for AQS-Hg²⁺ and AQS-CN⁻ complexes, respectively. The sensor AQS demonstrated practical applicability in detecting Hg²⁺ in real-world samples, including water, soil, and seafood, using PVA-coated gloves. The probe AQS successfully sensed mercury ions in <i>E. coli</i> bacterial cells, as visualized through laser confocal scanning microscopy, showcasing its potential for biological applications.</p>