Abstract Amino acids containing the thiol group play significant roles in keeping biological oxidation reduction balance. Due to the important cellular function of biothiols, it is imperative to develop efficient methods to detect biothiols in complicate biological systems. In this work, we developed a cyanine‐based fluorescent probe utilizing thioether as a detecting group, which showed the fluorescent "turn‐on" response towards GSH and it can be used as a biomarker to determine the cellular GSH.