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In this paper, a photoinduced electron transfer (PET)& twisted intramolecular charge transfer (TICT)-based fluorescent probe (1) for detecting biothiols (GSH/Cys/Hcy) and hydrogen sulfide with fluorescence turn on was developed. The probe could recognize hydrogen sulfide over primary ions and selectively detect GSH/Cys/Hcy over other amino acids with fluorescence turn-on (an ESIPT process). H 2 S can be distinguished from GSH/Cys/Hcy with wavelength shift by UV–Vis spectra. In addition, detection limits for H 2 S/GSH/Cys/Hcy of probe 1 were 1.42 μM (0–100 μM), 0.13 μM (0–40 μM), 0.27 (0–30 μM), 0.22 μM (0–40 μM), respectively. The proposed thiolysis of the 2,4-dinitrochlorophenyl ether reaction in identification process was verified by the characteristic peak in 1H NMR and HRMS spectra. Finally, the biological imaging experiments and low cytotoxicity investigations in HeLa cells demonstrated that probe 1 could provide a promising method for the determination of H 2 S and biothiols in aqueous solution and living cells. Unlabelled Image • A PET&TICT-based fluorescent probe 1 for H 2 S and GSH/Cys/Hcy has been developed. • Probe 1 possesses a high selectivity toward H 2 S and GSH/Cys/Hcy over amino acids. • The detection limit of probe 1 for H 2 S was 1.42 μM (in a range of 0–100 μM). • The CDL of probe 1 toward GSH/Cys/Hcy were 0.13 μM, 0.27 μM, 0.22 μM, respectivly. • The application of probe 1 for H 2 S imaging in HeLa cells was also achieved. [ABSTRACT FROM AUTHOR]