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[Display omitted] • An "off–on" long-wavelength fluorescent probe is synthesized for Cys detection. • The probe has a large Stokes shift (211 nm) and reacted with Cys specifically. • The probe can monitor Cys fluctuations caused by redox imbalance in HepG2 cells. • The probe can be used for fluorescence imaging in cells and mice. In this research, an "off–on" long-wavelength fluorescent probe (DCMN-Cl) based on (E)-2-(3-(2-(6-hydroxynaphthalen-2-yl)vinyl)-5,5-dimethylcyclohex-2-en-1-ylidene) malononitrile (DCMN) is designed and synthesized for cysteine (Cys) detection. DCMN-Cl exhibits a large Stokes shift (211 nm) and shows rapid response and high specificity to Cys. The fluorescence initensity at 635 nm reveals a good linear relationship with Cys concentration in the 0 to 50 μM range, and the detection limit is as low as 159 nM. The probe is also used for fluorescence imaging of Cys in cells and mice. Moreover, the probe provided visual evidence of Cu2+ and curcumin-induced intracellular Cys fluctuations. [ABSTRACT FROM AUTHOR]