Abstract: Screening on lactic acid bacteria with antibacterial activity, isolated from fermented pickle in eastern Inner Mongolia, was tested for Escherichia coli and Staphylococcus aureus as the indicator, and its antibacterial spectrum was tested.The optimum separation conditions of antibacterial substances were determined by single factor and L₉ ( 3⁴) orthogonal test, and detected antibacterial effect in commercial cattle feed.The result showed that the strain S1-4 had strong and stable antibacterial activity in 14 strains of lactic acid bacteria, and the diameter of inhibition zone of strain S1-4 to Escherichia coli and Staphylococcus aureus was ( 19.69 ± 0.71) mm and ( 20.59 ± 0.26) mm, respectively, and showed broad antimicrobial spectrum. The optimal extraction conditions for the antibacterial substances from strain S1-4 were as follows: the precipitation of p H was 5, the concentration of ethanol was 95%, the settling time was 8 h, and the ratio of material to liquid was 1 ∶ 4 ( v ∶ v) . In these conditions, the diameter of the inhibition zone was 39.53 mm, and increased by 23.18%.The fermentation broth of strain S1-4was added to the cattle feed, which could inhibit the growth of bacteria and mould in feed, and with the increase of the dosage, the inhibitory effect increased obviously.