<p>A new Schiff-base fluorescent probe namely methyl 2-(((2-hydroxypyridin-3-yl) methylene) amino) pyrimidine-5-carboxylate (HL) has been designed and synthesized. Structural characterization of the purely synthesized compound was acquired by IR spectroscopy, ¹H NMR, ¹³C NMR and Mass spectra. The Schiff base compound was explored as chemosensor for various transition as well as alkali metal ions viz. (Na⁺, K⁺, Ca²⁺, Ba²⁺, Sr²⁺, Cd²⁺, Ni²⁺, Co²⁺, Cu²⁺, Mn²⁺, Mg²⁺, Zn²⁺ and Fe³⁺). The study clearly reveals that the probe HL can rapidly and selectively recognize Mg²⁺, with the detection limit (LOD) of 7.2 × 10^− 7 M. The results of fluorescence spectroscopic studies clearly reveals that a remarkable enhancement of the fluorescence intensity of the probe HL was observed in presence of Mg²⁺ ions, which was barely impacted by other metal cations. The stoichiometric ratio of HL to Mg²⁺ was determined to be 1:1 from Job’s plot, B-H plot, ¹H NMR titration, Mass spectra and the binding constant was calculated as 3.7 × 10⁴ M⁻¹ using the Benesi-Hildebrand method. Reversibility nature of the complex has been studied in presence of EDTA, which in turn regenerated free ligand for further Mg²⁺ sensing. HL further exhibits INHIBIT logic gate behaviour with Mg²⁺ and EDTA as inputs, as observed from the emission response at 414&#xa0;nm. Over a broad pH range (9–12), this chemosensor shows excellent fluorescence sensing abilities to Mg²⁺. The probe HL has also been utilized for the detection of Mg²⁺ in real water samples. In addition, HL showed moderate antibacterial activity against <i>Escherichia coli</i> and <i>Staphylococcus aureus</i>, highlighting its bioactive potential.</p>