Experiments were conducted to evaluate the probiotic effect of bio-augmented Bacillus tequilensis AP BFT3 on improving production, immune response, and proteomic changes of Penaeus vannamei reared in a biofloc system. Penaeus vannamei larvae (PL13) were stocked in 100-L tanks at a rate of 100 no per tank to study the effect of B. tequilensis AP BFT3 with and without biofloc (BFT-PRO and PRO). Control tanks devoid of probiotic strain were maintained in a clear water system. The growth and survival considerably increased in probiotic added biofloc reared shrimp than probiotic added clear water reared ones and control. Water quality significantly improved in probiotic added (PRO) and biofloc-probiotics (BFT-PRO) system than control. Microbiological investigations indicate increased heterotrophic bacterial load in BFT-PRO compared to the PRO and control. The quality of the isolated microbes was analyzed in terms of enzyme production, and an abundance of enzyme-producing bacterial population was observed in BFT-PRO shrimp. Immune-related genes were significantly upregulated in BFT-PRO shrimp, followed by the PRO and control. The proteomic data (2D gel electrophoresis and MALDI-TOF) of muscle tissue from the experimental animals identified 11 differentially expressed proteins. The Daxx OS and Lit v 1 tropomyosin was found upregulated in BFT-PRO shrimps. Downregulation of Na⁺/K⁺ATPase was observed in biofloc with probiotic-supplied groups. The findings revealed that the BFT system’s efficacy could be improved through the addition of probiotics. The addition of B. tequilensis AP BFT3 as a probiotic in biofloc induced the expression of essential proteins, reducing contracting diseases during culture.