γ-Glutamyltranspeptidase (GGT) is a cell surface-associated enzyme, which has been proven to be closely related to many diseases. Thus, development of simple and effective GGT-activatable fluorescent probes for early diagnosis of diseases is of great significance. Herein, a series of simple isomeric pyridine-based GGT-activatable fluorescent probes has been successfully designed and synthesized. The preliminary experimental results indicate that Py-GGT-1 has a significant fluorescence response to GGT via a rare fluorescence-off approach. Further fluorescence response experiments, such as the time-/dose-dependent fluorescence change and the selectivity/specificity estimation, reveal that Py-GGT-1 has an excellent ability to monitor endogenous GGT activity. Mechanism studies through HRMS analysis, DFT calculations and molecular docking have theoretically verified the catalytic process between Py-GGT-1 and GGT. To develop its practical application, live-cell imaging and serum-sample analysis were carried out, demonstrating that Py-GGT-1 coud effectively track GGT within cells and organisms. Based its high selectivity and reasonable sensitivity, we expect that the probe will be applied in clinical diagnosis of GGT-related diseases in the future.