Beta-glucuronidase (βG) is a potential biomarker for cancer diagnosis and prodrug therapy. The ability to image βG activity in patients would assist in personalized glucuronide prodrug cancer therapy. However, whole-body imaging of βG activity for medical usage is not yet available. Here, we developed a radioactive βG activity-based trapping probe for positron emission tomography (PET). We generated a 124I-tyramine-conjugated difluoromethylphenol beta-glucuronide probe (TrapG) to form 124I-TrapG that could be selectively activated by βG for subsequent attachment of 124I-tyramine to nucleophilic moieties near βG-expressing sites. We estimated the specificity of a fluorescent FITC-TrapG, the cytotoxicity of tyramine-TrapG, and the serum half-life of 124I-TrapG. βG targeting of 124I-TrapG in vivo was examined by micro-PET. The biodistribution of 131I-TrapG was investigated in different organs. Finally, we imaged the endogenous βG activity and assessed its correlation with therapeutic efficacy of 9-aminocamptothecin glucuronide (9ACG) prodrug in native tumors. FITC-TrapG showed specific trapping at βG-expressing. CT26 (CT26/mβG) cells but not in CT26 cells. The native TrapG probe possessed low cytotoxicity. 124ITrapG preferentially accumulated in CT26/mβG but not CT26 cells. Meanwhile, micro-PET and wholebody autoradiography results demonstrated that 124I-TrapG signals in CT26/mβG tumors were 141.4-fold greater than in CT26 tumors. Importantly, Colo205 xenografts in nude mice that express elevated endogenous βG can be monitored by using infrared glucuronide trapping probes (NIR-TrapG) and suppressed by 9ACG prodrug treatment. 124I-TrapG exhibited low cytotoxicity allowing long-term monitoring of βG activity in vivo to aid in the optimization of prodrug targeted therapy.