<p>In this work, a new thiourea-based compound, TH4, was synthesized and its structure was investigated through FTIR, ¹H NMR and DFT analyses. The sensing properties of TH4 were systematically assessed against a wide range of metal ions to determine its selectivity and sensitivity. Among these, TH4 exhibited a pronounced fluorescence “turn-on” response selectively in the presence of Hg²⁺, indicating a strong and specific interaction. The formation of a stable TH4–Hg²⁺ complex was supported by Job’s plot analysis, which revealed a 1:2 binding stoichiometry. The sensor demonstrated excellent sensitivity with a detection limit of0.0036 ppm and a quantification limit of 0.012 ppm, enabling trace-level mercury detection. To assess its practical utility, TH4 was applied to the detection of Hg²⁺in environmental water samples including tap, pond, river, and lake water) and biological fluids (blood serum and urine). The sensor delivered high recovery rates ranging from 91.0 ± 0.22% to 104.8 ± 0.41%, confirming its reliability in real-world conditions. Additionally, both the free ligand and its Hg²⁺ complex displayed notable antimicrobial activity against selected bacterial and fungal strains. These findings demonstrate the dual-functional capability of TH4 as an effective fluorescent probe for mercury ion sensing and a potential antimicrobial agent, underscoring its significance in environmental analysis and biomedical research.</p>