To elucidate the effect of transglutaminase (TG) on the rheological properties of wheat gluten, this study investigates the underlying mechanisms by analyzing changes in gluten structure. The results demonstrated that the TG-treated gluten samples had higher storage modulus (G') and loss modulus (G") compared to the control, conversely, creep and recovery strains followed an opposite trend. Notably, the most pronounced effects were observed with adding 2 U/g TG for 20-30 min. Size exclusion/reversed phase-high performance liquid chromatography profiles revealed that the treatment with TG elevated the levels of glutenin subunits, alongside reduced α- and γ-gliadins, promoting gluten aggregation. Moreover, the extractability of gluten gradually decreased due to TG-induced oxidation of sulfhydryl groups, which formed new disulfide bonds and cross-linked products. This structural modification reduced surface hydrophobic regions and promoted the aggregation of low molecular weight proteins into larger molecular weight aggregates. Microstructural analysis further confirmed that TG enhanced gluten network stability through covalent cross-linking. Overall, this study demonstrates that TG enhances the rheological characteristics of wheat gluten by facilitating the formation of a more robust network structure, driven by cross-linking reactions and disulfide bond formation.