Abstract: Saccharomyces cerevisiae and Lactobacillus brevis were selected to conduct sourdough fermentation in this work. The changes of structural and physicochemical properties of wheat starch during the fermentation were studied, so as to provide theoretical reference and support for improving the quality of sourdough products as well as the mechanism of starch modification by fermentation. Five fermentation time points at 3, 6, 9, 12, and 24 hours were selected during Saccharomyces cerevisiae (Sce) fermentation and Saccharomyces cerevisiae mixed with Lactobacillus brevis (Sce-Lbr) fermentation. Structural and physicochemical properties of wheat starch extracted by washing and centrifugation during sourdough fermentation were analyzed. The results showed that:the surface morphology of starch samples was significantly changed by fermentation. The granules showed serious erosions with the increase of fermentation time, and the erosions mainly occurred in large A-starch granules. Sce and Sce-Lbr fermentation did not show significant differences in the surface morphology of the granules. The amylose content in both fermentation methods decreased significantly from 0 to 3 h at the initial fermentation stage, suggesting that the amylose was broken down into low-molecular carbohydrates, which remained stable as the fermentation continued. In addition, Sce-Lbr fermentation decreased more amylose in the starch. During Sce fermentation, the solubility of starch decreased significantly with the increase of fermentation time, while the swelling power did not change significantly. The solubility of starch in Sce-Lbr fermentation during 12 h decreased significantly with the increase of fermentation time, and then increased significantly after 24 h. Both methods did not change the starch crystalline structure. The relative crystallinity of starch increased significantly with the increase of fermentation time during Sce fermentation. In detail, this value increased from 20.70% to a maximum of 27.87% after 24 h fermentation, indicating that the amorphous regions of starch granules were gradually damaged during fermentation. While in Sce-Lbr fermentation, it increased to a maximum of 25.28% after 12 h fermentation but then decreased slightly to 24.41% after 24 h fermentation, suggesting that the crystalline regions may also be slightly destroyed as the fermentation continues after the destruction of the starch amorphous regions. The enthalpy of starch increased with the increase of fermentation time during Sce fermentation, however, a fluctuating upward trend appeared in Sce-Lbr fermentation. Overall, the results of our work showed that the granular structure of wheat starch was altered during sourdough fermentation, leading to the changes of physicochemical properties.