Abstract: Short-chain fatty acids (SCFAs) are fatty acids with a carbon chain of fewer than six atoms, primarily generated through the fermentation of dietary fiber by gut microbiota. Owing to their antibacterial, anti-inflammatory, immunomodulatory, and glycemic-regulating properties, SCFAs hold considerable promise for use as food preservatives and dietary supplements. To enhance SCFA production in a sustainable and cost-effective manner, this study employed agro-industrial byproducts-namely wheat bran and wheat starch wastewater-as a composite fermentation substrate. The medium composition and fermentation parameters were systematically optimized using the strain Lactobacillus paracasei EG9. Initially, single-factor experiments assessed the influence of feed-liquid ratio, exogenous carbon sources, nitrogen sources, and inorganic salts on SCFA yield. Subsequently, the effects of fermentation time, temperature, inoculation volume, initial pH, and shaker speed were evaluated. Based on these results, a Box-Behnken design coupled with response surface methodology (RSM) was applied to model and optimize the key interactive factors: fermentation time, temperature, and inoculation volume. The optimal medium formulation comprised a 1∶8 g/mL feed-liquid ratio supplemented with 0.6% resistant starch, 1% yeast extract, and 0.4% K₂HPO₄. The optimal fermentation conditions were identified as: 36 h duration, 37 ℃, 5% inoculation volume, initial pH of 6.0, and shaker speed of 150 r/min. Under these optimized conditions, the total SCFA concentration reached 2.539 g/L, representing a 2.02-fold increase relative to pre-optimization levels. In conclusion, the refined medium composition and fermentation parameters substantially improved the capacity of Lactobacillus paracasei EG9 to produce SCFAs, thereby enabling efficient and high-value biotransformation of wheat bran and wheat starch wastewater.