Relevant studies have demonstrated that abnormal concentrations of cysteine (Cys) will directly affect normal biol. processes within cells, leading to various diseases.As one of the important intracellular organelles, the Golgi apparatus (GA) serves as the primary site for protein synthesis.Cysteine is involved in protein formation and metabolism and other activities in cells, so its concentration is directly related to the proper function of the GA.Therefore, the development of a highly selective and sensitive method to detect the concentration of cysteine in the Golgi apparatus has important biomedical significance for diagnosing diseases.In this study, a naphthalimide-dicyanoisophorone conjugate was utilized as the fluorescent unit, benzenesulfonamide as the Golgi-localizing group, and acrylate as the recognition moiety to construct a Golgi-targeted near-IR probe for Cys detection.When Cys was absent, the intramol. charge transfer (ICT) effect was suppressed, resulting in minimal fluorescence emission from the probe.However, upon the addition of Cys, the ICT process was restored, inducing an enhancement in the NIR fluorescence emitted by the probe.Within the level scope from 0.2 μM to 10 μM, the fluorescence intensity of the probe demonstrated a linear correlation with the level of Cys.Furthermore, the limit of detection was determined as 0.024 μM.The fluorescent probe for Cys demonstrated excellent selectivity, superior sensitivity, rapid response time, and a broad pH working scope.Besides, this probe exhibited minimal cytotoxicity and was successfully applied for the detection of Cys in the GA of live cells.Furthermore, the probe was also effective in detecting cysteine in zebrafish and mice.