To investigate impacts of short-clustered maltodextrin (SCMD), trehalose (TH), and guar gum (GUAR) on mediating stabilizations of gluten proteins against cold denaturation, we focused on conformational transformations of gluten protein during frozen storage and its molecular mechanism. Firstly, we found SCMD markedly improved hydration features and decreased the surface hydrophilicity of gluten proteins, with the integrity and continuity of gluten network improving after 8-week frozen storage, compared to TH and GUAR. Furthermore, SCMD and TH addition hindered the conversion of α-helix to disordered β-fold and β-turn, while GUAR did not. Similar outcomes were observed in results of amino acid microenvironment. Molecular mechanism mining indicated the SCMD could establish additional hydrogen bonds with water and protein molecules, suppressing cold denaturation of protein. Our findings found saccharide composition and size were important factors in mediating protein stabilization during frozen storage, which will offer novel insights into the mitigation of cold-induced protein denaturation.