The phenothiazine-based fluorometric chemosensor, N′,N‴-((1E,1′E)-(10-ethyl-10H-phenothiazine-3,7-diyl)bis(methaneylylidene))bis(4-methylbenzenesulfonohydrazide) (L), has been meticulously designed, synthesized, and evaluated for its capabilities in metal ion detection. This sensor exhibits a remarkable selectivity for Cu2+ and Fe3+ ions, showcasing its potential applicability over a spectrum of other common metal cations in acetonitrile (CH3CN) solution. The interaction with Cu2+ and Fe3+ results in a distinctive fluorescence on–off response. Impressively low detection limits of 5.54 × 10−9 M for Cu2+ and 3.11 × 10−9 M for Fe3+ have been achieved, attesting to the high sensitivity of the sensor. The recognition mechanism of L towards Cu2+ and Fe3+ has been systematically investigated through various analytical approaches. Job plot measurements, Fourier-transform infrared (FT-IR) titration, and high-resolution mass spectrometry (HRMS) have provided insightful details regarding the binding stoichiometry and nature of the interactions between L and the target metal ions. Beyond fundamental characterization, the practical utility of the probe has been demonstrated in the bio-imaging of Cu2+ and Fe3+ in a mouse fibroblast cell line (NIH3T3), underscoring its potential for biological applications. Additionally, the sensor has been successfully employed for the quantification of Cu2+ and Fe3+ in real samples, showcasing its versatility in environmental monitoring. The results collectively highlight the efficacy of L as a robust chemosensor with significant potential for various analytical and biomedical applications, contributing to the growing repertoire of metal ion detection methodologies.