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This research centers on the development and synthesis of a longwave fluorescence probe, labeled as <b>60T</b>, designed for the simultaneous detection of hydrogen sulfide, cysteine/homocysteine, and glutathione. The probe showcases a swift response, good linearity range, and heightened sensitivity, boasting that the detection limits of the probe for Cys, Hcy, GSH and H₂S were 0.140, 0.202, 0.259 and 0.396 μM, respectively. Notably, its efficacy in monitoring thiol status changes in live MCF-7 cells is underscored by a substantial decrease in fluorescence intensity upon exposure to the thiol trapping reagent, N-ethyl maleimide (NEM). With an impressive red emission signal at 630 nm and a substantial Stokes shift of 80 nm, this probe exhibits remarkable sensitivity and selectivity for biothiols and H₂S, indicating promising applications in the diagnosis and surgical navigation of relevant cancers.