γ-Glutamyl transpeptidase (GGT), a type of cell membrane-bound enzyme, is closely involved in a wide range of physiological and pathological processes, and a large number of fluorogenic probes have been developed to detect the activity of GGT. However, the use of these imaging reagents to visualize GGT activity in vivo is largely limited because of rapid diffusion and clearance of activated fluorophores. Herein, by merging quinone methide and a fluorogenic enzyme substrate, we report an activatable self-immobilizing near-infrared probe for the in vitro and in vivo imaging of GGT activity. This probe is initially fluorescently silent, but the selective activation by GGT is able to significantly increase its fluorescence intensity at 714 nm and covalently anchor activated fluorophores at the site of interest. We have shown that this probe induced a much stronger fluorescence on live GGT-overexpressing cells compared to regular fluorogenic probes and allowed wash-free and real-time imaging of enzyme activity. More importantly, the use of this probe in the imaging of GGT activity in U87MG tumor-bearing mice by i.v. administration indicates that this self-immobilizing reagent is capable of efficiently enhancing its retention at the detection target and thus leads to much improved detection sensitivity compared to regular fluorogenic probes. This study demonstrates the advantage of fluorogenic probes with activatable anchors in the noninvasive imaging of enzyme activity in highly dynamic in vivo systems.