A novel styrylbenzimidazolium dye containing a maleimide group 5 was synthesized and characterized using proton nuclear magnetic resonance spectroscopy and mass spectrometry. The photophysical properties [ultraviolet–visible (UV–vis) light absorption and fluorescence spectra, fluorescence quantum yield, and fluorescence lifetime] were investigated. Spectroscopic characterization of the novel styrylbenzimidazolium‐based dye under various conditions is presented and its usefulness to detect biothiols proved. The addition of biothiols [l‐cysteine (l‐Cys), l‐homocysteine (l‐Hcy), l‐glutathione (l‐GSH)] to compound 5 in phosphate buffer (0.1 M, pH 7.4) containing 10% CH3CN induced a 15–28‐fold enhancement in fluorescence intensity at 410 nm. The limits of detection of compound 5 for l‐Cys, l‐Hcy, and l‐GSH were estimated as 0.114, 0.118, and 0.059 μM, respectively. Evaluation of the cytotoxicity of 5 using the PrestoBlue assay for HeLa cells was also determined. The examined compound revealed a slight cytotoxicity against HeLa cells under experimental conditions.