Bread wheat (Triticum aestivum L.) is one of the crucial cereals consumed by human beings and wheat gluten, the natural macro molecules, mainly determines the processing quality of wheat dough. The high-molecular-weight gluten in subunits (HMW-GSs) of gluten proteins are recognized as one of the main components regulating the rheological properties of dough. The over expressed Bx7 subunit (Bx7OE) has been reported to improve wheat quality and rheological properties of dough, however its effect on secondary and micro- structures of gluten is still unclear. In this study, we evaluated the composition of main storage proteins in wheat grains of two near isogeniclines and studied the effect of Bx7 subunit expression level on the secondary structures of gluten and micro-structure of gluten during dough mixing process. Results showed the protein content, HMW-GSs proportion in total gluten ins and free sulfhydryl content increased in the flour of HMW-Bx7OE wheat line, and the accumulation of unextractable polymeric protein during grain filling stage accelerated. It was found that the content of β-sheets in secondary structures of gluten increased and a more compact micro-structure of gluten network formed in the dough. Protein network analysis characterized and quantified the alterations in the gluten micro-structure. In the process of dough mixing, protein area, total protein length, number of junctions and branching rate reach the peak at dough development time, which was consistent with Chopin mixing profile.Interestingly, during dough mixing, the above-mentioned parameters of HMW-Bx7OE showed less changes than those of HMW-Bx7 wheat line, indicating Bx7OE improved the dough stability during mixing. To conclude, Bx7O Ealters the secondary and micro- structures of gluten and thus improves the mixing and rheological properties of wheat dough.