This experiment investigated the changes of the gluten protein structure of the dough during manual kneading. Results: At the first 2 minutes of kneading, the free sulfhydryl group in the dough was decreased by 10.69%, the disulfide bond was increased by 4.48%, and then was slowly increased or decreased; the hydrogen bonds was increased significantly (P＜0.05), and the hydrophobic force was decreased significantly (P＜0.05); the random coil in the secondary structure had an initial sharp drop of 56.52%, and the β-sheet had an initial increase of 32.34%, followed by a small increase or decrease, and the α-helix and β-turn have no significant changes; The SDS extractable rates of glutenin all decreased to varying degrees; the peak area of B/C-LMW-GS increased by 4.56%, 2.61%, 4.11%, 6.73%, 3.18%, 0.81%, and the peak area of α-gliadins decreased by 22.42%, 10.87%, 11.59%, 12.87%, 9.22%, and 4.35%, respectively. Hand kneading promoted the cross-linking polymerization reaction of gluten protein and made its protein conformation develop in a more orderly and stable direction, thereby enhanced the viscoelasticity of the dough.