Two new Schiff bases were synthesized from 1‐(2,4‐dihydroxyphenyl)ethanone and pyridine derivatives. Both compounds were characterized using infrared, UV–Vis., 1H NMR, 13C NMR and mass spectral studies. Density functional theory (DFT) calculations were performed for both the Schiff bases with 6‐31G(d, p) as the basis set. Vibrational frequencies calculated using the theoretical method were in good agreement with the experimental values. Both the Schiff bases were highly fluorescent in nature. The cation‐recognizing profile of the compounds was investigated in aqueous methanol medium. The Schiff base 4‐(1‐(pyridin‐4‐ylimino)ethyl)benzene‐1,3‐diol (PYEB) was found to interact with Fe(III) and Cu(II) ions, whereas the Schiff base 4,4′‐((pyridine‐2,3‐diylbis(azanylylidene))bis(ethan‐1‐yl‐1‐ylidene))bis(benzene‐1,3‐diol) (PDEB) was found to detect Cu(II) ions. The mechanism of recognition was established as combined excited state intramolecular proton transfer (ESIPT)–chelation‐enhanced fluorescence (CHEF) effect and chelation‐enhanced quenching (CHEQ) process for the detection of Fe(III) and Cu(II) ions, respectively. The stability constant of the metal complexes formed during the sensing process was determined. The limit of detection for Fe(III) and Cu(II) ions with respect to Schiff base PYEB was found to be 1.64 × 10−6 and 2.16 × 10−7 M, respectively. With respect to Schiff base PDEB, the limit of detection for Cu(II) ion was found to be 4.54 × 10−4 M. The Cu(II) ion sensing property of the Schiff base PDEB was applied in bioimaging studies for the detection of HeLa cells.