A simple fluorescent chemosensor 1 for the sequential detection of Hg²⁺ and cysteine or glutathione was developed by combination of thiosemicarbazide and 8-hydroxyjulolidine-9-carboxaldehyde. Sensor 1 exhibited an ‘ON–OFF’ fluorescence quenching response in the presence of Hg²⁺, which was explained by theoretical calculations. 1 also showed high selectivity in the presence of potential competitors such as Ag⁺ and Pb²⁺. Moreover, the resulting Hg²⁺-2·1 complex acted as an efficient ‘OFF–ON’ sensor for cysteine or glutathione, showing recovery of 2·1 from Hg²⁺-2·1 complex. Therefore, sensor 1 can be employed as a practical fluorescent chemosensor for recognition of Hg²⁺ and thiol-containing amino acids in aqueous solution.