Abstract: The best culture conditions and antibacterial ability of one strain of Enterococcus faecalis(H_1-1-2), which was isolated from koumiss collected from farms in Inner Mongolia-Xilinguole region, were studied. The antibacterial factor from the culture medium of the bacteria were also extracted and identified. The results indicate that the best temperature is 37℃ for culturing Enterococcus faecalis, the pH value begin to decrease in 4 h of incubation, the bacteria entered the phase of log growth in 7 h, the antibacterial factor is found in medium in 8 h and reaches the highest level in 12 h. After analysis with super-filtration, gel column chromatography, thin layer chromatography, Legend of UV absorb, the factor is a more than 50 ku protein-like substance. The factor can maintain its antibacterial ability in the range of pH 1.0 to pH 10.0, it is durable to heat in pH 4.0, 60℃ for 30 min and some surfactants, but it is damaged easily by protease and catalase.