A novel fluorescent probe bearing triphenylamine, thiophene and thiosemicarbazide (TPA-TSC) was developed, and its distinct features were investigated. TPA-TSC demonstrated excellent selectivity towards Hg2+ ions, with a limit of detection (0.14 nM) among the lowest reported to date. It also exhibited a turn-off response by forming stable complex for a brief period (<30 s). Job plot analysis and high-resolution mass spectrometry (HRMS) revealed that TPA-TSC bound to Hg2+ with a 2:1 stoichiometry in an aqueous environment, remarkably the probe successfully detected Hg2+ ions in seabass, swordfish and different samples of water. In all, TPA-TSC offers a promising method for accurately determining the quality of seafood and water samples polluted with Hg2+.