Abstract: Lactobacillus casei (Lb. casei) plays an important role in food biological preservation. In order to study the separating method of organic acids and antibacterial activity of Lb. casei, the culture supernatant of Lb. casei was extracted by ethyl acetate. The extraction phases were concentrated by the rotary evaporator. The antibacterial activity was determined by modified spot-on-lawn method. The main organic acids of extraction were determined by high performance liquid chromatography (HPLC). The result showed that the ethyl acetate cyclosis appeared very good antibacterial activity against the three kinds of foodborne pathogens, Escherichia coli O157:H7, Staphylococcus aureus and Listeria monocytogenes. Inhibition of the growth of Escherichia coli O157:H7 and Listeria monocytogenes were most obvious, the diameters of inhibition zone were (20.00±0.82) mm and (21.67±0.94) mm, respectively. The main antibacterial organic acids were lactic acid, acetic acid and phenyl lactic acid analyzed by HPLC. The study provided a feasible method for isolating the antibacterial substances from the culture supernatant of lactic acid bacteria.