A completely water-soluble, high quantum yield blue-fluorescent benzimidazole derivative (AQ), containing a rigid benzimidazole-thiophene structure, was synthesized. Among 21 metal ions, the fluorescence of AQ was selectively turned off by Cu²⁺ to form an AQ-Cu²⁺ ensemble. Thereafter, the fluorescence of the AQ-Cu²⁺ ensemble was turned on by sulfide (S²⁻) with high selectivity and sensitivity in pure water solution. In comparison with AQ-Ag⁺ and AQ-Hg²⁺ ensembles, AQ-Cu²⁺ was the only ensemble that was capable of detecting a sulfide anion. Also, the fluorescence intensity of AQ was linearly proportional to the concentration of Cu²⁺ and S²⁻. Both Cu²⁺ and S²⁻ were detected within a minute in vitro. Moreover, AQ worked best in the pH range of 5–10 and had a limit of detection of 50 nM and 354 nM for Cu²⁺ and S²⁻ respectively. It was employed for the detection of sulfide in human lung cancer A549 cells with low cytotoxicity.