Abstract: Bacillus with inhibiting Escherichia coli function was screened from the soil. The fermentation medium and fermentation conditions were optimized by single factor and orthogonal test.The results showed that Bacillus G3E, which had strong bacteriostatic effect against Escherichia coli was screened and identified as Bacillus amyloliquefaciens subsp.plantarum. The best fermentation medium for G3E was starch 10 g/L, calcium chloride 4 g/L, yeast extract 4 g/L, tryptone 4 g/L, urea 2 g/L.The optimum fermentation conditions were as follows:The fermentation temperature was 40℃, the fermentation time was 40 h, the pH value was 7.0.At this time, the diameter of the bacteriostasis circle was (25.5±0.5) mm.