To elucidate the mechanisms underlying the changes in the rheological properties of dough made from wheat flour during maturation, the molecular structure of gluten before and after maturation was characterized. Wheat flour was matured under three distinct conditions for predetermined durations. The development time, stability, and maximum force of dough peaked at 7.10 min, 8.58 min, and 88.98 N, respectively, after 40 days of maturation at 25 degrees C and 40 degrees C. Compared to the control, the storage modulus of dough made from wheat flour matured at 40 degrees C increased, while creep compliance decreased, indicating improved deformation resistance and a closer resemblance to viscoelastic solid materials. SDS-PAGE and molecular weight distribution indicated that maturation induces the binding of gluten peaks, evoking small molecular weight proteins to form larger protein clusters through folding. Compared to the control, the content of disulfide bonds significantly (P < 0.05) increased, tightening the protein network, while fluorescence intensity decreased after 40-50 days. This is accompanied by a distinct cross-linkage structure, confirmed by AFM. Among the three maturation conditions, 40 degrees C had the most pronounced effect, followed by 25 degrees C. This study offers insights and a theoretical basis for adjusting maturation conditions to enhance wheat flour quality.