Abstract <p>This study describes the radiosynthesis and preclinical testing of ⁸⁹Zr-labeled conjugated durvalumab, a new immuno-PET probe aimed at improving in vivo stability and facilitating sensitive imaging of PD-L1 dynamics. Durvalumab was conjugated site-specifically to the bifunctional chelator 3,4,3-LI(1,2-HOPO) (LI-HOPO) and radiolabeled using ⁸⁹Zr under mild conditions. MALDI-TOF-MS and SDS-PAGE characterization validated successful conjugation with the chelator : antibody ratio of ~5 : 1 and the antibody integrity maintained. The radiolabeling efficiency reached 70.45 ± 1.8, with radiochemical purity &gt;98.5%. The immunoreactivity, determined by Lindmo assay against PD-L1-positive CT26 cells, was 85.4 ± 1.2%. The radiotracer was highly serum stable in vitro (&gt;96% intact at 120 h). PET/CT imaging in CT26 tumor-bearing mice revealed progressively higher tumor uptake, with the highest value at 120 h post injection. The tumor uptake correlated with the PD-L1 expression and immune modulation, particularly in mice receiving combination therapy with durvalumab and the epigenetic modulator I-BET762. Ex vivo biodistribution established the increased PD-L1 expression in tumors with high radiotracer accumulation. [⁸⁹Zr]Zr-3,4,3-LI(1,2-HOPO)-durvalumab is a promising immuno-PET tracer with high radiochemical stability, high specificity, and efficient tumor targeting. It permits quantitative noninvasive evaluation of PD-L1 expression and immune modulation, exhibiting significant potential as a new radiotracer for the guidance and monitoring of cancer immunotherapy.</p>