Abstract: Wheat bran is rich in diverse bioactive constituents, among which wheat bran α-amylase inhibitors (WBAI) have garnered extensive research attention owing to their prominent potential in regulating postprandial glycemia and developing functional foods for metabolic health. This study systematically explored wheat bran as a natural WBAI resource, optimized its extraction conditions, and performed comprehensive structural and functional characterization of WBAI. Multiple extraction parameters were investigated to determine the optimal preparation protocol. The molecular-weight distribution of purified WBAI fractions was analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and their structural features and functional groups were identified using Fourier-transform infrared spectroscopy (FTIR). Furthermore, the pH and thermal stability of WBAI were evaluated, and enzymatic kinetic assays were conducted to elucidate the inhibitory mechanism of WBAI against α-amylase. The results showed that the optimal extraction conditions were a solid-liquid ratio of 1∶5 mg/mL, 0.10 mol/L NaCl, an extraction duration of 90 min, and 70% ammonium sulfate saturation. Under these optimized conditions, scale-up extraction using 100 g of wheat bran yielded 1.26 g of lyophilized WBAI powder, with a protein content of 813 μg/mL, inhibitory activity of 107.2 U/mL, specific activity of 131.9 U/mg, and a total extraction yield of 43%. Stability assays revealed that WBAI retained stable inhibitory activity at pH 6-7 and at temperatures between 40 and 70 ℃. Kinetic analysis confirmed that WBAI inhibits α-amylase via a reversible competitive mechanism. Collectively, these findings verify that wheat bran is a reliable and sustainable raw material for the preparation of high-activity α-amylase inhibitors. The optimized extraction process and systematic structural and functional characterization provide a solid theoretical basis for the industrial development and application of WBAI, facilitating the incorporation of WBAI into functional food formulations and realizing the high-value utilization of cereal by-products.