Abstract: Lactobacillus helveticus strain AJT, isolated from traditional fermented dairy samples in Inner Mongolia, China, its antibacterial substance’s production increased continuously from 6h, and reached a maximum after 20h. Obtained through ethyl acetate and methanol extraction, the antibacterial substance, had strong antibacterial activity against Gram - positive bacteria such as Staphylococcus aureus, Gram - negative bacteria such as Escherichia coli and fungi such as Penicillium expansum after eliminating the effect of organic acids and hydrogen peroxide. The active principle was proteinaceous in nature or a mixed substance since the inhibitory activity decreased after treated with trypsin, pepsin, protease K and papain. The inhibitory activity was stable after heating at different temperature for 30min and over the pH range from 2. 0 to 5. 0, UV light did not affect the activity of the substance, which indicated the antimicrobial substance produced by AJT belong to bacteriocin-like substance.