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Background Immunotherapy has emerged as a promising avenue for malignant tumors treatment, but methods to validate its in vivo antitumor activity remain limited. Granzyme B, a key mediator of immune-mediated cell death, is a potential biomarker for assessing immunotherapy efficacy. This study aimed to develop a radiotracer with high sensitivity and specificity for in vivo imaging of granzyme B.Methods 10 granzyme B targeting inhibitors were synthesized and evaluated to improve binding affinity. The most promising compound was conjugated with 1,4,7-triazacyclononane-N,N',N"-triacetic acid chelator and radiolabeled with Al 18F to generate radiotracer 18F-G1. Its imaging and pharmacokinetic properties were compared with those of the recently developed radiotracer 68Ga-grazytracer. 18F-G1 was applied to assess T cell-mediated and natural killer (NK) cell-mediated antitumor immunity. In addition, the cyclic guanosine monophosphate AMP synthase-stimulator of interferon genes (cGAS-STING) pathway-targeted therapies were also quantitatively evaluated using 18F-G1.Results 18F-G1 was produced with high radiochemical purity of >98% through a simple radiolabeling procedure. It displayed a 4.2-fold improvement in granzyme B binding affinity compared with grazytracer. In the anti-programmed cell death protein-1 antibody-treated MC38 tumor model, 18F-G1 outperformed 68Ga-grazytracer with significantly higher tumor uptake (2.1-fold increase in maximum tumor uptake) and superior tumor-to-muscle contrast (7.46 vs 2.33). For NK cell-based therapy, 18F-G1 positron emission tomography (PET)/CT successfully monitored granzyme B release following NK-92MI cell injection, with peak signals observed 24 hours postinjection. 18F-G1 PET/CT detected robust immune activation in SR-717-treated tumors compared with other cGAS-STING pathway-targeted drug treatments, which correlated with the highest CD8+ T cell infiltration, granzyme B expression level and tumor growth inhibition.Conclusions 18F-G1 is an effective granzyme B imaging radiotracer. Its superior imaging properties make it a promising tool for non-invasively evaluating the therapeutic efficacy of various types of immunotherapies with high sensitivity.