Positron emission tomography (PET) is a molecular imaging technique that visualizes pathophysiology in the body using radiotracers at tracer doses (~μg). PET would provide the information regarding not only pharmacokinetics of radiolabeled compounds, but also target engagements, patient selection, and biomarkers. Previously small molecules are widely used as radiotracers, but recently biopharmaceuticals are launched, providing a novel type radiotracer. In general, antibodies are radiolabeled by long physiological half-lives radionuclides such as Cu-64 and Zr-89 because of their slow pharmacokinetics. However, shorter half-lives radiolabeled tracers (C-11 and F-18) might be suitable on the point view of radiation. Now small protein ligands such as affibodies (~7 kDa) are developed as a radiotracer. We are trying to develop a novel approach to label proteins for PET imaging, which are based on radiolabeled amino acids and cell-free protein synthesis system. In this review, we introduced the topics of protein-based PET tracers.