Abstract: Amino acid water-soluble fertilizer boasts advantages such as full water solubility, high activity, and rich nutritional content. It serves to enhance crop yield and quality, while also contributing to soil pH balance and soil improvement. As a novel and functional fertilizer, it employs a mixture of rapeseed meal and soybean meal as raw materials. Utilizing laboratory-preserved Lactobacillus plantarum G71, commercial yeast, and Bacillus subtilis, the fermentation process is optimized through methodologies like orthogonal testing, Plackett-Burman experiments, climbing response surface methodology, and Box-Behnken Design. The optimal fermentation conditions were found to be a fermentation time of 217.68 hours, initial pH of 9.12, material-to-water ratio of 1:7.008, inoculation rate of 3%, and turning interval of 24 hours. Verified results indicate a yield of free amino acids reaching 76.8 g·kg^-1 within the fermentation solution. Therefore, employing mixed bacterial fermentation effectively enhances the amino acid content of fermentation solutions derived from agricultural residues like rapeseed meal and soybean meal, subsequently elevating resource utilization efficiency. This approach holds significant implications for the advancement of ecologically sustainable, high-value agriculture.