Positron Emission Tomography (PET) is a non-invasive molecular imaging technique for clinical diagnostic with the detection of pathological lesions. Small radioactive molecules such as fluorodeoxyglucose have been mainly used as PET tracers. However, they have some challenges in the aspect of their specificity. Recently, small protein ligands (6-7 kDa), affibody molecules, are attracting increasing interest as an innovative PET tracer owing to their promised high specificity and affinity. In this study, we propose a novel radiolabeling method for protein with F-18, termed TAG-encoded cell-free protein synthesis system with F-18 labeled amino acid, to use affibody molecules as PET tracers. Furthermore, we show the successful preparation of radiolabeled affibody molecules targeted to HER2 and PD-L1 with this method. A small animal PET imaging with F-18 labeled affibodies surely demonstrated the accumulation of tracers in tumors expressing HER2 or PD-L1. This technique will be a powerful tool for the evaluation of potential candidates for PET tracers in preclinical settings because of its facile radiolabeling of protein just by adding the template DNA plasmids.