Biological thiols, especially low molecular weight thiols, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play a pivotal role in physiological and pathological systems. Thus, the detection of biothiols is highly important for early diagnosis of diseases and evaluation of disease progression. Herein, we developed a highly selective and sensitive ratiometric fluorescent 8-Cl BODIPY-based probe with high fluorescence quantum yields. The probe displayed a sensitive response to Cys and Hcy over other biothiols, which can be visualized colorimetrically and/or fluorescently. The probe was successfully applied to detect Cys in human plasma, demonstrating its great value for practical application in biological sample analysis.