Pretargeted concept in positron emission tomography (PET) together with bioorthogonal chemistry is an elegant solution to study processes with slow pharmacokinetics by utilizing radiotracers labeled with short-lived radionuclides. Namely, radiotracers based on tetrazine ligation with trans-cyclooctene (TCO) via the inverse electron demand Diels–Alder (IEDDA) reaction have become a state-of-the-art for the pretargeted PET imaging. For radiolabeling of tetrazine scaffolds, indirect radiofluorination methods are often preferred, as tetrazines are vulnerable to harsh conditions typically necessary for the direct radiofluorination. ¹⁸F-Fluoroglycosylation is an indirect radiofluorination method, which allows the introduction of a widely accessible glucose analog 2-[¹⁸F]fluoro-2-deoxy-d-glucose ([¹⁸F]FDG) to aminooxy-functionalized precursors via oxime formation. Here, we report the biological evaluation of [¹⁸F]FDG-Tz as a tracer for pretargeted PET imaging of TCO-functionalized molecular spherical nucleic acids (MSNA) against human epidermal growth factor receptor 2 (HER2) mRNA. The oxime ether formation between [¹⁸F]FDG and tetrazine oxyamine resulted in [¹⁸F]FDG-Tz with high radiochemical purity (>99%) and moderate yields (6.5 ± 3.6%, n = 5). Biological evaluation of [¹⁸F]FDG-Tz in healthy mice indicated favorable pharmacokinetics with quick blood clearance, urinary excretion as the main elimination route, and the absence of GLUT1 transportation. The successful pretargeted experiments with TCO-functionalized MSNA revealed higher tumor uptake compared to preclicked MSNA in HER2-expressing human breast cancer xenograft-bearing mice.