This study presents the synthesis and application of a novel fluorescent probe, NR-ClO, for the detection of hypochlorite ion (ClO–) in biological systems. The probe was synthesized through a nucleophilic substitution reaction between Nile red and dimethylcarbamothioic chloride. The synthesized probe had high sensitivity and selectivity towards ClO–, with a detection limit of 75 nM and a linear range of 0.1–200 μM. The probe's efficacy was validated through in vitro studies using HepG2 cells and in vivo experiments using a mouse model of rheumatoid arthritis. The findings demonstrate that the NR-ClO probe is a promisingly reliable tool for real-time monitoring of ClO– in complex biological environments.