Inclusion of microbial fermented soybean meal in broiler feed has induced advantageous outcomes for their performance and gastrointestinal health by exhibiting probiotic effects. In this study, soybean meal (SBM) was subjected to double-stage microbial fermentation utilizing functional metabolites of fungi and bacteria. In the broiler diet, DFSBM replaced SBM at 0%, 25%, 50%, and 100%. DFSBM was reported to have higher protein content and total essential, nonessential, and free amino acids (increased by 3.67%, 12.81%, 10.10%, and 6.53)-fold, respectively, compared to SBM. Notably, phytase activity and lactic acid bacteria increased, while fiber, lipid, and trypsin inhibitor contents decreased by 13.82%, 38.31%, and 73.50%, respectively, in a diet containing 100% DFSBM compared to SBM. Improved growth performance and apparent nutrient digestibility, including phosphorus and calcium, and pancreatic digestive enzyme activities were observed in groups fed higher DFSBM levels. In addition, higher inclusion levels of DFSBM increased blood immune response (IgG, IgM, nitric oxide, and lysozyme levels) and liver antioxidant status. Jejunal amino acids- and peptide-transporter-encoding genes (LAT1, CAT-1, CAT-2, PepT-1, and PepT-2) were upregulated with increasing levels of DFSBM in the ration. Breast muscle crude protein, calcium, and phosphorus retention were increased, especially at higher inclusion levels of DFSBM. Coliform bacteria load was significantly reduced, while lactic acid bacteria count in broiler intestines was increased with higher dietary levels of DFSBM. In conclusion, replacement of SBM with DFSBM positively impacted broiler chicken feed utilization and boosted chickens’ amino acid transportation, in addition to improving the nutritional value of their breast meat.