Abstract: To address the low utilization rate of wheat germ resources and insufficient bioactivity of wheat embryo globulin (WEG), this study took strain combination, solid-liquid ratio, and fermentation time as single factors. After screening key parameters via single-factor experiments, an L₉(3⁴) orthogonal experiment was used to optimize the fermentation process. The optimal conditions were determined as the strain combination of Lactobacillus acidophilus and Acetobacterium (1∶1), a solid-liquid ratio of 1∶10 g/mL, and a fermentation time of 18 h. These optimal conditions were then applied to modify WEG. Structural changes of fermented globulin (GFWG) were characterized by Fourier transform infrared spectroscopy (FT-IR), circular dichroism (CD), scanning electron microscopy (SEM), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The activity mechanism of GFWG was analyzed through in vitro antioxidant assays (DPPH and hydroxyl radical scavenging rates) and free amino acid composition, while its in vivo efficacy was evaluated using a cyclophosphamide-induced immunosuppressed mouse model. Results showed that under the optimal process conditions, the DPPH radical scavenging rate of GFWG reached 73.78% at 10 mg/mL, which was significantly higher than that of unfermented WEG. Structurally, GFWG exhibited an 72.20% increase in β-sheet content, a 44.19% decrease in α-helix content, and its surface transformed from sheet-like aggregates to a porous fibrous network. Among free amino acids, histidine and methionine increased by 148.30% and 200.00%, respectively, while leucine and valine showed more significant increases (619.40% and 267.70%, respectively). Animal experiments confirmed that GFWG increased the hepatic activities of superoxide dismutase (SOD) and glutathione (GSH) activities in model mice by 42.60% and 37.80%, respectively, and reduced the spleen index from 8.60 to 5.56 (a decrease of 35.35%), which was close to the normal group value of 4.86. This study demonstrates that by optimizing the three factors of strain combination, solid-liquid ratio, and fermentation time, mixed fermentation with Lactobacillus acidophilus and Acetobacterium can induce WEG structural reconstruction, enhancing its antioxidant and immune protective functions. Thus, this study provides a theoretical and technological basis for the high-value utilization of plant proteins.