Bacterial infections are considered to be a major cause of mortality in yellow catfish (Pelteobagrus fulvidraco) culture. Using probiotic bacteria to control potential pathogens has been developed and accepted as a good approach in aquaculture. Bacillus has potential advantages as probiotics because it can antagonize intestinal pathogenic bacteria, improve the ability of disease prevention, produce enzymes and vitamins, and its spores has strong tolerance to heat, pressure, acid, alkali, enzymes and other adverse environments. This paper focused on selection and characterization of Bacillus which would be used as potential probiotics in aquaculture. Sixty five spore-producing bacterial strains were got from intestinal tract of Pelteobagrus fulvidraco by using water bath kettle at 80℃. Among of them, three strains (F13, F14 and F15) which had better antagonistic activity against commonly pathogenic bacteria of fish (Cytophaga columnaris, Edwardsiella tard, Aeromonas hydrophila, Aeromonas sobria and Escherichia coli) were obtained by Oxford- cup tests. The diameter of inhibition zone was up to 9.1—14.1 mm (Oxford-cup diameter was 6 mm). The three strains were identified as Bacillus subtilis based on physiological and biochemical (API 50CHB) tests referring to data bank of Biomerieux. The strain F14 which had the strongest inhibiting activity was chosen and measured temperature and acid tolerance, antibacterial activity of metabolites, viability in feed after keeping in different conditions. The results showed that survival of F14 were 96.7%, 95.3% and 52.97% after cells were heated in 80, 90 and 100 ℃ ℃ ℃ for 5 minutes respectively. The cell number declined rapidly in 90℃ and 100℃ with time. When being heated in 100℃ for 10 minutes, survival was 8.3% only. From pH values 3.0 to 5.0, its viability was more than 98% and 87% after being treated for 1 or 2 hours. It was suggested that the viability and stability of F14 would not be destroyed by feed mixing and pelletizing, and strong acid gastric juice. The metabolites of F14 filtered by porous membrane showed high antibacterial activities against fish pathogen (A. hydrophila). The number of A. hydrophila declined rapidly in metabolites of F14 after 2 hours, and none survived cells were detected after 24 hours. One of the reason was antimicrobial substances in spent culture supernatant secreted by F14 inhibit pathogenic bacteria. The livability of probiotics in feed was very important for saving and applying. The viability of the bacteria in the diets was assessed following storage of the diet at room temperature, 4 and ?20℃ for 20 days. F14 viability showed no significantly difference in the feed stored at either 4 or ?20℃ after 20 days of storage. In conclusion, F14 had credible stability and wider activity against pathogens of fish. It might be used as potential probiotics in aquaculture.