摘要： Thiols play a vital role in both the physiological process and organic synthesis field, including aliphatic thiols (e.g., Cys, Hcy, and GSH) and thiophenols. Due to the similarity of thiols in terms of molecular structure and chemical properties, it is difficult for conventional fluorescent probes to distinguish them, which hinders the progress of biological and pathological research. Keeping this in mind, a dual-site and dual-excite fluorescent probe (YY) was designed to distinguish Cys, Hcy, and thiophenols by three different reaction paths. When excited at 470 nm, YY only exhibits a fluorescence OFF-ON response towards thiophenols. However, when excited at 453 nm, YY not only displays a fluorescence OFF-ON response towards Hcy and thiophenols (λem = 499 and 561 nm) but also presents a two-stage fluorescence response towards Cys, which possesses a fluorescence OFF-ON response in the first stage (λem = 501 nm) and then a fluorescence ON-OFF response in the second stage (λem = 556 nm). This specific fluorescence response indicates that YY has ability to overcome the above-mentioned challenge to achieve discriminative detection of Cys, Hcy, and thiophenols qualitatively, which promotes the study of thiols in the field of physiology and pathology. Furthermore, cell-imaging studies show that YY can be applied to the imaging of exogenous Cys, Hcy, and thiophenols through two different emission channels.