Abstract Background Fatty acid binding protein 3 (FABP3) is a target with clinical relevance and the peptide ligand ACooP has been identified for FABP3 targeting. ACooP is a linear decapeptide containing a free amino and thiol group, which provides opportunities for conjugation. This work is to develop methods for radiolabeling of ACooP with fluorine-18 (¹⁸F) for positron emission tomography (PET) applications, and evaluate the binding of the radiolabeled ACooP in human tumor tissue sections with high FABP3 expression. Results The prosthetic compound 6-[¹⁸F]fluoronicotinic acid 4-nitrophenyl ester was conveniently prepared with an on-resin ¹⁸F-fluorination in 29.9% radiochemical yield and 96.6% radiochemical purity. Interestingly, 6-[¹⁸F]fluoronicotinic acid 4-nitrophenyl ester conjugated to ACooP exclusively by S-acylation instead of the expected N-acylation, and the chemical identity of the product [¹⁸F]FNA-S-ACooP was confirmed. In the in vitro binding experiments, [¹⁸F]FNA-S-ACooP exhibited heterogeneous and high focal binding in malignant tissue sections, where we also observed abundant FABP3 positivity by immunofluorescence staining. Blocking study further confirmed the [¹⁸F]FNA-S-ACooP binding specificity. Conclusions FABP3 targeted ACooP peptide was successfully radiolabeled by S-acylation using 6-[¹⁸F]fluoronicotinic acid 4-nitrophenyl ester as the prosthetic compound. The tissue binding and blocking studies together with anti-FABP3 immunostaining confirmed [¹⁸F]FNA-S-ACooP binding specificity. Further preclinical studies of [¹⁸F]FNA-S-ACooP are warranted.