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Rheumatoid arthritis (RA) is a long-term systemic inflammatory disease that causes severe joint pain. Golgi stress caused by redox imbalance significantly involves in acute and chronic inflammatory diseases, in which cysteine (Cys), as a representative reducing agent, may be an effective biomarker for RA. Hence, in order to achieve RA early detection and drugs evaluation, based on our previous work about innovative Golgi-targeting group, we established a phenylsulfonamide-modified fluorescence probe, Golgi-Cys, for the selective fluorescence imaging of Cys in Golgi apparatus in vivo. By application of Golgi-Cys, the Cys changes under Golgi stress in cells were elucidated. More importantly, we found that the probe can be effectively utilized for the RA detection and treatment evaluation in situ.