The cultural microbiomes of 27 bacteria colonies were isolated from Mugil cephalus for analysis of the antibacterial and antagonistic activities. A potent probiotic bacterium was characterized using16S r RNA sequencing. The potent strain was added to fish diet to perform the challenge test and to study the growth and immunological parameter. The extracellular proteins from the probiotic were collected and characterized using MALDI TOF/TOF. Out of G27, G9 strain inhibited all the five pathogenic strains. An isolated bacterium was identified as Bacillus subtilis PRBD09 with accession number KF765648. After 35 days of feeding period B. subtilis PRBD09 enhance the both cellular and humoral immune responses, which responsible for survive of the Mugil cephalus against Aeromonas hydrophila infection. The MALDI TOF sample 08 and 09 were recognized as hypothetical proteins based on the MALDI TOF sample. A cytidinedeaminase was found in samples 10, 11, and 12. Extracellular proteins may be involved for the immunological increase in Mugil cephalus against Aeromonas hydrophila, according to the current research.