An efficient thiocarbohydrazide based fluorescencesensor S5 for the selective detection of Hg2+ in semi-aqueous medium has been successfully designed and synthesized. The absorption (UV-Vis) and emission (fluorescence) properties of S5 were studied in various solvents. The sensor S5 displayed no distinct changes in absorption with various solvents; however some remarkable changes were noticed in fluorescence studies. Solvent dependent spectral characterization of S5 was explored via the Lippert-Mataga plot. UV-Vis and fluorescence studies also revealed that the sensor S5 could selectively detect Hg2+ ion over the other tested metal ions. The interaction of S5 with Hg2+ leads to colour change from colourless to yellow. The observed quenching in fluorescence intensity may be due to chelation enhanced fluorescence quenching (CHEQ). The sensing mechanism was arrived through DFT and 1H NMR titration studies. The limit of detection was found to be 1.26 nM with a wide pH range of 4–10. Moreover, sensor S5 could demonstrate sensing efficiency for lower concentrations of Hg2+ in real sample analysis. Its performance, as a test strip and in analysis of various water samples conferred its superior selectivity. © 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim