A turn-on fluorescent probe for the detection of cysteine (Cys), an essential amino acid in biological systems, is developed based on Cys-mediated O-phenyl benzothioate native chemical ligation (NCL) for the first time. Cys plays important roles in many physiological processes, thus the development of Cys monitoring methods has been highlighted in fundamental research field. We prepared an on-off type fluorescent probe, CysP-1, which showed highly sensitive and selective response towards Cys. Furthermore, the probe showed superior sensing ability for Cys in the cancer cells with low cytotoxicity, and released hydrogen sulfide (H2S). This successful demonstration will encourage further Cys-related applications in a variety of fields.