Faster and more efficient approaches for radiolabeling biomolecules with short-lived 18F are in dire need. Herein we report a new 18F-labeled prosthetic group containing an acetylene function that permits the labeling of biomolecules via click chemistry. This template, propargyl 4- [18F]fluorobenzoate ([18F]PFB) was synthesized from a quaternary salt precursor in decay-corrected radiochemical yields of 58 ± 31%. Several model compounds containing an azide moiety — benzyl azide, two lysine derivatives and a transglutaminase-reactive peptide — were labeled using [18F]PFB via a click reaction in decay-corrected radiochemical yields of 88 ± 4%, 79 ± 33%, 75 ± 5%, and 37 ± 31%, respectively. Our results suggest that the novel agent [18F]PFB is a potentially useful template for the 18F-labeling of biomolecules via click chemistry.