Protein tyrosine kinase-7 (PTK7), a member of receptor tyrosine kinase superfamily initially identified as colon carcinoma kinase-4, is highly expressed in various human malignancies. Its expression was found to correlate with aggressive biologic behaviors such as increased cell proliferation, invasiveness, and migration. Despite the importance and unmet need of imaging PTK7 in vivo, there is currently no clinically relevant method to visualize tumoral PTK7 expression noninvasively such as PET or SPECT. This study aimed to develop a specific, selective, and high-affinity PET radioligand based on single-stranded DNA aptamer to address this challenge. Sgc8, a 41-oligonucleotide that targets to PTK7, was labeled with (18)F using a 2-step radiochemical synthesis, which featured a direct 1-step radiofluorination on the distinctive spirocyclic hypervalent iodine(III) precursor to give (18)F-fluorobenzyl azide followed by copper-mediated click conjugation with Sgc8-alkyne. (18)F-Sgc8 was evaluated in vitro and in vivo in 2 cell lines, HCT116 and U87MG, which express high and low amounts of PTK7, respectively. Sgc8 was labeled efficiently with (18)F in an isolated radiochemical yield of 62% ± 2%, non-decay-corrected based on (18)F-fluorobenzyl azide. (18)F-Tr-Sgc8 was found to possess high-affinity binding to both cell lines, with binding affinity values of 2.7 ± 0.6 nM for HCT116 and 16.9 ± 2.1 nM for U87MG. In vivo PET imaging clearly visualized PTK7 expression in HCT116 xenografted mice, with tumor uptake of 0.76 ± 0.09 percentage injected dose per gram (%ID/g) at 30 min after injection for the subcutaneous tumor model and greater than 1.5 %ID/g for the liver metastasis model. U87MG xenograft tumors had much lower tracer accumulation (0.13 ± 0.06 %ID/g at 30 min after injection), which was consistent with the lower expression of PTK7 in this tumor model. The labeled aptamer was rapidly cleared from the blood through the kidneys and bladder to give high tumor-to-blood and tumor-to-muscle ratios of 7.29 ± 1.51 and 10.25 ± 2.08, respectively. The (18)F-radiolabeling methodology shown here is a robust procedure for labeling aptamers and similar chemical moieties and can be applied to many different targets. Quantification of PTK7 using (18)F-Tr-Sgc8 may be suitable for clinical translation and might help in the future to select and monitor appropriate therapies.