Abstract: This study utilized corn steep liquor (CSL), a byproduct of corn processing, as the raw material. Koji with high protease activity was employed to ferment and degrade CSL proteins, and fermentation conditions were optimized using response surface methodology to investigate the effect of fermentation on its antioxidant activity. The results showed that the optimal process parameters were: Koji addition (based on protease activity) of 28 U/mL, temperature of 53 °C, fermentation time of 5 h, and pH of 5.7. Under these conditions, the soluble protein content in fermented CSL reached 137.83±2.07 mg/mL, representing an 8.81% increase compared to unfermented CSL. Fermentation promoted the hydrolysis of proteins in CSL and increased the content of small peptides, reduced the average particle size of CSL proteins, and significantly increased the absolute value of Zeta potential (P<0.05). After fermentation, the relative content of α-helix in the secondary structure of CSL proteins decreased by 4.59%, while β-sheet increased by 2.82%, and β-turn increased by 1.77%. Scanning electron microscopy (SEM) revealed that the macromolecular proteins in CSL were degraded into fragments with folded and porous surface structures. At a concentration of 5 mg/mL, the DPPH radical scavenging rate of fermented CSL was 51.35 %±2.06%, the ABTS⁺ radical scavenging rate was 92.23%±1.57%, and the FRAP value was 38.54±1.78 µmol/L, representing increases of 11.85%, 7.16%, and 21.77%, respectively, compared to the unfermented group. This study opens a new pathway for the high-value utilization of corn byproducts and the development of novel plant-based protein resources.