We designed a two-step-release system that enables protein release from hydrogel in response to glucose. In the system, one of the heterodimeric coiled-coil forming peptides was initially fixed to a hydrogel via a boronate-diol interaction. The peptide was specifically released from the hydrogel by externally incoming glucose. As a result, a protein immobilized in another hydrogel was librated owing to the displacement by the peptide at its hydrogel-bound coiled-coil moiety.