<p>This study discusses a Schiff base chemosensor ((E)-N’-(2-hydroxybenzylidene)isonicotinohydrazide (HISN)), specifically designed for the rapid detection of Cu²⁺ and Ni²⁺ ions in aqueous medium. HISN characterization was conducted using various methods, including FT-IR, HRMS, ¹H NMR, ¹³C NMR, fluorometric techniques, and UV-Visible spectroscopy. In an acetonitrile solution (1 µM, pH = 7.4), HISN exhibited a noticeable and significant color change from colorless to yellow when exposed to Cu²⁺ and Ni²⁺ ions. The UV-visible absorption spectrum of the HISN-Cu²⁺ and HISN-Ni²⁺ complexes displayed both bathochromic (red) and hypsochromic (blue) shifts. Additionally, the presence of Cu²⁺ and Ni²⁺ ions notably increased the fluorescence intensity of HISN. The limits of detection (LOD) and quantification (LOQ) for HISN with respect to Cu²⁺ and Ni²⁺ ions were established. The LOD values are 3.394 × 10^− 9 M for Cu²⁺ and 1.646 × 10^− 7 M for Ni²⁺, while the corresponding LOQ values are 1.131 × 10^− 8 M for Cu²⁺ and 5.487 × 10^− 7 M for Ni²⁺. Data analysis from Job’s plot, along with HRMS and FT-IR spectral data, suggested a 1:1 stoichiometric relationship between HISN and both Cu²⁺ and Ni²⁺ ions. Further, the complex formation between HISN-Cu²⁺ and HISN-Ni²⁺ was confirmed by Density Functional Theory (DFT) calculations using Gaussian 16 software. Geometry optimization of these complexes is carried out using the exchange-correlation functional B3PW91 and the basis set 6-311 + G(d, p). MTT assay results indicated that HISN formulations are cytocompatible and do not harm cells at varying concentrations. Furthermore, HISN has been utilized as a fluorescent imaging probe for intracellular Cu²⁺and Ni²⁺ ions.</p>