Abstract: Response surface methodology was used to optimize the conditions for producing bacteriocin, biological methods was applied for localization of bacteriocin and the stabilization of bacteriocin of Lactobacillus acidophilus. Results showed that the optimal bacteriocin production for Lactobacillus acidophilus were: 38 ℃, p H7.0 and 18 hours in MRS medium. Lactobacillus acidophilus fermentation broth could inhibit the growth of Escherichia coli which was mainly in the extracellular extracts. The bacteriocin had good heat stability under 40~70 ℃.The inhibitory activity was decreased by the treatment with proteinase K, pepsin and trysin.The active p H of the bacteriocin to the pathogen was from 3.0 to 10.0.The inhibitory activity was stable with the time of ultraviolet irradiation 0.25~1 h. These results may provide an important reference for separation and application of bacteriocin produced by Lactobacillus acidophilus.