To determine the probiotic potential of bacterial isolated from fish gut, which enhance the immune system of the host animal and improve the microbial balance of the digestive tract. Fish samples were collected from the trodden vaigai river in Theni district. Seventeen bacterial cultures were isolated from fish gut .Among these, only six bacterial cultures are considered as a probiotic depending on the biochemical and molecular characterization technique. The probiotic characterization tests for acid tolerance, bile salt tolerance, antimicrobial activity and antibiotic test profiles were also determined. The probiotic endosymbiotic bacterial spp prevailing in selected freshwater fishes, namely Oreochromis mossambicus and Labeo rohita .were isolated and characterized by using the 16s rRNA sequencing method. Among the 17 bacterial strains isolated from fish gut samples, 6 bacterial strains were found to be probionts which was confirmed by various probiotic analysis tests. These probionts showed both antibacterial activity and antibiotic susceptibility. Two probiotic bacterial spp such as B. cereus (KU167636) and B. subtilis (KU167639) were selected, based on their beneficiary activities. The overall completed study revealed that the isolated Bacillus spp. fulfills the required criteria for probiotic such as tolerance to harsh conditions such as low pH and high bile salt concentration and it can be produce bacteriocin extra-cellularly which inhibits pathogenic organisms. These isolates were used for potential probiotics.