When the cell environment changes or is stimulated, the Golgi apparatus will respond to the corresponding stress, through the opening of related pathways, the expression of corresponding substances can be promoted or inhibited to achieve the purpose of controlling cell redox homeostasis and reducing cytotoxicity. Intuitive analysis of the changes in the content of various substances in the process of stress has important guiding value for the further study of stress response, drug evaluation and clinical diagnosis. Therefore, for the Cys overexpressed during the oxidative stress of the Golgi apparatus, we developed a specific and sensitive fluorescent probe (Gol-NCS) to visually monitor the biologically important Cys in real time. The probe has low cytotoxicity and shows great potential in cell and zebrafish imaging, it can detect the changes of endogenous and exogenous cysteine. It is important to explore the synthetic pathway of Cys during Golgi stress by using the Golgi targeting performance of the probe Gol-NCS. It is confirmed by fluorescence imaging for the first time that the activity of CSE enzyme plays a decisive role in the formation of Cys. Therefore, probe Gol-NCS with excellent photochemical properties is expected to provide help for the research on the involvement of Cys in Golgi stress.