Positron emission tomography is the imaging modality of choice when it comes to the high sensitivity detection of key markers of thrombosis and inflammation, such as activated platelets. We, previously, generated a fluorine‐18 labelled single‐chain antibody (scFv) against ligand‐induced binding sites (LIBS) on activated platelets, binding it to the highly abundant platelet glycoprotein integrin receptor IIb/IIIa. We used a non‐site‐specific bio conjugation approach with N‐succinimidyl‐ 4‐[18F]fluorobenzoate (S[18F]FB), leading to a mixture of products with reduced antigen binding. In the present study, we have developed and characterised a novel fluorine‐18 PET radiotracer, based on this antibody, using site‐specific bio conjugation to engineer cysteine residues with N‐[2‐(4‐ [18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM). ScFvanti‐LIBS and control antibody mut‐scFv, with engineered C‐terminal cysteine, were reduced, and then, they reacted with N‐[2‐(4‐ [18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM). Radiolabelled scFv was injected into mice with FeCl3‐induced thrombus in the left carotid artery. Clots were imaged in a PET MR imaging system, and the amount of radioactivity in major organs was measured using an ionisation chamber and image analysis. Assessment of vessel injury, as well as the biodistribution of the radiolabelled scFv, was studied. In the in vivo experiments, we found uptake of the targeted tracer in the injured vessel, compared with the non‐injured vessel, as well as a high uptake of both tracers in the kidney, lung, and muscle. As expected, both tracers cleared rapidly via the kidney. Surprisingly, a large quantity of both tracers was taken up by organs with a high glutathione content, such as the muscle and lung, due to the instability of the maleimide cysteine bond in vivo, which warrants further investigations. This limits the ability of the novel antibody radiotracer18F‐scFvanti‐LIBS to bind to the target in vivo and, therefore, as a useful agent for the sensitive detection of activated platelets. We describe the first fluorine‐18 variant of the scFvanti‐LIBS against activated platelets using site‐specific bio conjugation.