A novel fluorescent “off” sensor, R-β-D-1, was obtained in high yield (91.2%) by using octahydronaphthol as a backbone, introducing an alkyne group at the 2-position, and linking azido-glucose via a click reaction. The sensor was analyzed by scanning electron microscopy and transmission electron microscopy and was found to be a self-assembled vesicle. AFM results showed that the fluorescence burst was extinguished by the addition of Fe³⁺, and the fluorescence was restored by the addition of cysteine. This is due to charge transfer within the molecular structure, resulting in the ICT effect and phototransfer of electrons (PET), as well as redshifting (from 331 nm to 351 nm) and quenching of the fluorescence. The self-assembled vesicles of the fluorescent sensor R-β-D-1 encapsulated Fe³⁺, but upon addition of cysteine, the vesicles of R-β-D-1–Fe³⁺ were also complexed with it, forming the R-β-D-1–Fe³⁺–L-Cys complex, at which point fluorescence gradually returned. Therefore, the fluorescence test of this probe showed that the lowest detection limit of iron ions was 1.67 × 10⁻⁷ mol L⁻¹, and its complexation mode was in the form of 1 + 1. The novel probe formed by R-β-D-1–Fe³⁺ can be used for the fluorescence detection of cysteine.