Claudin18.2 (CLDN18.2) is a tight junction protein that is overexpressed in a variety of solid tumors such as gastrointestinal cancer and oesophageal cancer. It has been identified as a promising target and a potential biomarker to diagnose tumor, evaluate efficacy, and determine patient prognosis. TST001 is a recombinant humanized CLDN18.2 antibody that selectively binds to the extracellular loop of human Claudin18.2. In this study, we constructed a solid target radionuclide zirconium-89 (89Zr) labled-TST001 to detect the expression of in the human stomach cancer BGC823CLDN18.2 cell lines. The [89Zr]Zr-desferrioxamine (DFO)-TST001 showed high radiochemical purity (RCP, >99%) and specific activity (24.15 ± 1.34 GBq/μmol), and was stable in 5% human serum albumin, and phosphate buffer saline (>85% RCP at 96 h). The EC50 values of TST001 and DFO-TST001 were as high as 0.413 ± 0.055 and 0.361 ± 0.058 nM (P > 0.05), respectively. The radiotracer had a significantly higher average standard uptake values in CLDN18.2-positive tumors than in CLDN18.2-negative tumors (1.11 ± 0.02 vs. 0.49 ± 0.03, P = 0.0016) 2 days post injection (p.i.). BGC823CLDN18.2 mice models showed high tumor/muscle ratios 96 h p.i. with [89Zr]Zr-DFO-TST001 was much higher than those of the other imaging groups. Immunohistochemistry results showed that BGC823CLDN18.2 tumors were highly positive (+++) for CLDN18.2, while those in the BGC823 group did not express CLDN18.2 (-). The results of ex vivo biodistribution studies showed that there was a higher distribution in the BGC823CLDN18.2 tumor bearing mice (2.05 ± 0.16 %ID/g) than BGC823 mice (0.69 ± 0.02 %ID/g) and blocking group (0.72 ± 0.02 %ID/g). A dosimetry estimation study showed that the effective dose of [89Zr]Zr-DFO-TST001 was 0.0705 mSv/MBq, which is within the range of acceptable doses for nuclear medicine research. Taken together, these results suggest that Good Manufacturing Practices produced by this immuno-positron emission tomography probe can detect CLDN18.2-overexpressing tumors.© 2023 The Authors.