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Benzimidazole-based compound 2-(<i>p</i>-tolyl)-1<i>H</i>-benzo[d]imidazole (<b>3</b>) and its derivative probe <b>A-B</b> have been synthesized for the highly selective detection and quantification of Cys in human serum. The photophysical properties of <b>A-B</b> and compound <b>3</b> were evaluated by UV-vis absorption and fluorescence spectroscopy. <b>A-B</b> showed high selectivity and sensitivity for Cys among tested analytes, including amino acids, anions, and cations. <b>A-B</b> selectively reacts with Cys and results in compound <b>3</b> with fluorescence turn-on effect. <b>A-B</b> did not show any interference from the components in the serum matrix for Cys detection in the human serum sample. <b>A-B</b> detects Cys in serum samples with 2.3–5.4-fold better LOD than reported methods. The detection limit of 86 nM and 43 nM in HEPES buffer using UV-visible and fluorescence spectroscopy, respectively, makes <b>A-B</b> an excellent chemosensor for Cys detection.