γ-Glutamyl transpeptidase (GGT) is one of the biomarker of cancer, hepatitis, and numerous other diseases. The accurate analysis of GGT is useful for the early diagnosis of these diseases. In this work, Probe 1, a ratiometric fluorescent probe based on 2,3,5,6-tetrafluoroterephthalonitrile, was designed for GGT detection. The results indicated that Probe 1 can sensitively and selectively detect GGT in phosphate buffered solution and complex biological systems (e.g., blood serum). Furthermore, Probe 1 has been successfully applied for ratiometric imaging of GGT in cancer cells and normal cells.