Biotin-PEG4-methyltetrazine is a biotinylated PEG derivative containing methyltetrazine moiety and a hydrophilic PEG spacer which increases water-solubility. This reagent can react with TCO-containing compounds to form a stable covalent bond without the catalysis of Cu or elevated temperatures. The inverse-electron demand Diels-Alder cycloaddition reaction of TCO with tetrazines is the fastest bioorthogonal reaction with exceptional selectivity.