γ-Glutamyltranspeptidase (GGT) is a cell -membrane-associated enzyme which has been recognized as a promising biomarker for the diagnosis of many malignant tumors. Herein, we rationally designed a fluorine-18 labeled small-molecule probe, [18F]γ-Glu-Cys(StBu)-PPG(CBT)-AmBF3 (18F-1G), by applying a biocompatible CBT-Cys condensation reaction and ingeniously decorating it with a GGT-recognizable substrate, γ-glutamate (γ-Glu), for enhancing PET imaging to detect GGT level of tumors in living nude mice. The probe had exceptional stability at physiological conditions, but could be efficiently cleaved by GGT, followed by a reduction-triggered self-assembly and formation of nanoparticles (NPs) progressively that could be directly observed by transmission electron microscopy (TEM). In in vitro cell experiments, 18F-1G showed GGT-targeted uptake contrast of 2.7-fold to that of 18F-1 for the detection of intracellular GGT level. Moreover, the higher uptake in GGT overexpressed HCT116 tumor cells (∼4-fold) compared to GGT-deficient L929 normal cells demonstrated that 18F-1G was also capable of distinguishing some tumor cells from normal cells. In vivo PET imaging revealed enhanced and durable radioactive signal in tumor regions after 18F-1G coinjecting with 1G, thus allowing real-time detection of endogenous GGT level with high sensitivity and noninvasive effect. We anticipated that our probe could serve as a new tool to investigate GGT-related diseases in the near future.