A novel fluorescent probe (AQDA) based on quinizarin is designed and synthesized. Owing to a nucleophilic addition and a specific intramolecular cyclization reaction, the probe displays high selectivity towards cysteine (Cys) relative to other natural amino acids. The maximum fluorescent intensity is 30-fold that of the initial value in the presence of 5.0 equiv. Cys, and its detection limit is 0.158 μM. The recognition mechanism is further confirmed through mass spectroscopy and proton nuclear magnetic resonance titration. Simultaneously, the fluorescence enhancement mechanism is characterized by theoretical calculations, and experimental data are consistent with the theoretical results. Finally, the cellular imaging experiment verifies that AQDA possesses the capacity to detect endogenous Cys in living cells.