Abstract: This paper aimed to investigate the release efficiency of peptide at carbon terminal triggered by tetrazine bioorthogonal click-to-release reaction, and further explored the potential application of this reaction in functional modification and mild cleavage in solid-phase peptide synthesis. Thirteen peptide derivatives modified by trans-cyclooctene (TCO) were designed and synthesized, which were reacted with tetrazine to release the peptides. The results showed that the release rates of peptide were 90.0% to 97.7% in one hour. The strategy has good compatibility with the functional side-groups and the length of peptides, which expands the applications scope of tetrazine bioorthogonal click-to-release reaction. At the same time, a novel bifunctional trans-cyclooctene molecule was designed and synthesized. The active peptide GIRLRG was modified by fluorophore on the solid-phase resin, and released through tetrazine click-to-release reaction under mild condition, providing a new strategy for the solid-phase modification and release strategy of the peptide.