In the present study, an attempt was made to isolate, identify, and characterize probiotic bacteria from the intestine of native freshwater fish Etroplus suratensis. Intestine of three fishes were dissected out, homogenized and serially diluted to 10-1-10-5 dilutions. Diluted samples were spread plated and 16 bacterial colonies showing morphological variations were sub-cultured. Out of which, seven bacterial strains which were able to grow in MRS (De Man, Rogosa and Sharpe) broth were evaluated for probiotic properties. Antimicrobial test was done by well diffusion method on the selected bacterial strains against Bacillus subtilis, Bacillus cereus, and Staphylococcus aureus. Four bacterial strains which showed antimicrobial properties were then tested for antibiotic sensitivity on ampicillin, chloramphenicol, erythromycin, tetracycline and vancomycin through disc diffusion method, followed by pH tolerance test at 5,6,7 and 8. The three bacterial strains which showed tolerance to varying pH were subjected to bile salt tolerance at concentrations, 0%, 0.15% and 0.30%. All the three strains were found to be tolerant to different bile salt concentrations and later tested for in vitro antioxidant activity using DPPH (2,2-Diphenyl 1-picryl hydrazyl) radical scavenging assay. The bacteria which showed more potent activity in all the experiments was subjected to gram staining and was found to be gram negative. Biochemical tests on the selected bacteria showed negative result for Indole, Methyl red, Voges Proskauer and Oxidase test and proved positive for Citrate test, TSI (Triple Sugar Iron) test, Mannitol motility test and Catalase test. The bacterium was identified as Enterobacter asburiae by molecular characterization using BLAST with 98.79% homology. The result of the above study opens the sustainable utilization of Etroplus suratensis gut as a probiotic source, which is otherwise considered as a waste product in aquaculture.