This study investigated the morphology distribution, molecular structure, and aggregative properties variation of gluten protein during dough mixing stage and interpreted the interaction between starch with different sizes and protein. Research results indicated that mixing process induced glutenin macropolymer depolymerization, and promoted the monomeric protein conversion into the polymeric protein. Appropriate mixing (9 min) enhanced the interaction between wheat starch with different particle sizes and gluten protein. Confocal laser scanning microscopy images showed that a moderate increase in B-starch content in the dough system contributed to forming a more continuous, dense, and ordered gluten network. The 50A-50B and 25A-75B doughs mixed for 9 min exhibited a dense gluten network, and the arrangement of A-/B-starch granules and gluten was tight and ordered. The addition of B-starch increased α-helixes, β-turns, and random coil structure. Farinographic properties indicated that 25A-75B composite flour had the highest dough stability time and the lowest degree of softening. The 25A-75B noodle displayed maximum hardness, cohesiveness, chewiness, and tensile strength. The correlation analysis indicated that starch particle size distribution could influence noodle quality by changing the gluten network. The paper can provide theoretical support for regulating dough characteristics by adjusting the starch granule size distribution.