Abstract: To explore the self-assembly gelation behavior of wheat gliadin peptides and develop novel plant-based functional gel materials, the targeted peptide segment was acquired via ultra-filtration through the controlled enzymatic hydrolysis of gliadin by alkaline protease in this study. A series of multi-scale characterization methods, including texture profile analysis, rheometry, scanning electron microscopy (SEM), atomic force microscopy (AFM), Zeta potential analysis, FT-IR spectroscopy, hydrophobicity determination, disulfide bond determination, and so on were comprehensively employed to assess its physico-chemical property, micro-structure and molecular mechanism. According to the findings, the molecular weight of gliadin peptide ranged predominantly from 1 to 5 kDa. This peptide segment could form a stable gel at a concentration of 15%, and its hardness increased with concentration. Its rheological behavior showed shear-thinning characteristics; SEM and AFM revealed that the gel featured regular porous structure and uniform nano-spheric morphology; Zeta potential analysis (−43.63±1.41 mV) indicated an excellent colloidal stability. In terms of molecular mechanism, FT-IR spectroscopy confirmed the hydrogen bonding as its primary driving force, with the increased α-helix content (25.1%→37.9%) representing the crucial conformational transition; hydrophobic interaction and disulfide bond exchange jointly promotes assembly; sequence analysis revealed typical amphiphilic arrangement patterns of (XZXZ)_n and (UCUC)_n. This study has innovatively elucidated the mechanism by which gliadin peptide forms a stable self-assembled gel via hydrogen-bond-dominated molecular forces. It provides both a theoretical basis and a material foundation for professionals to apply the plant peptide-based gel into food texture profile improvement and pharmaceutical delivery vector development.