Abstract: OBJECTIVE To optimize the gel separation system suitable for the separation of gekko small molecular peptides and to provide experimental basis for improving the bioavailability of gekko peptides and the separation and analysis of small molecular peptides. METHODS Biomimetic enzymatic hydrolysis was used to hydrolyze gekko protein in vitro, and folin-ciocalteu colorimetry was used to determine the changes in peptide content before and after biomimetic enzymatic hydrolysis. SDS-PAGE gel system and Tricine-SDS-PAGE gel system were respectively used to compare and analyze the changes in the number and molecular weight of peptide before and after biomimetic enzymatic hydrolysis. RESULTS After the gekko was treated by biomimetic enzymatic hydrolysis, the content of small peptides increased while the molecular weight decreased. Moreover, the number of bands separated by Tricine-SDS-PAGE gel system was significantly more than that of SDS-PAGE gel system. CONCLUSION The three-layer gel system of Tricine-SDS-PAGE electrophoresis is more suitable for the separation of gekko small molecular peptides.