The copper-free azide-alkyne cycloaddition was broadly applied in numerous research fields. Herein, we report a facile Cu-free click reaction utilizing fluoride-responsive azide and alkynyl pyridinium cycloaddition at ambient temperatures in aqueous media. The reactivity of alkynyl pyridinium was successfully masked by a silyl-protecting group at the alkyne group, and the deprotection could be readily achieved with the addition of F-, which renders the reactivity. The substrates were readily synthesized and proven to be stable at the bench. This bioorthogonal fluoride-responsive click reaction was then successfully employed in peptide modification, protein labeling, and cell imaging, suggesting its potential in various applications.