Abstract: Lactic acid bacteria(LAB) strains with strong antagonistic activity to Bacillus spp. were isolated from freshwater fish intestine in the study. The results showed that the diameters of inhibiting Bacillus cereus and Bacillus licheniformis by strain LY-19 from carp intestinal were 16.89 mm and 17.43 mm,respectively. The antagonistic activity was mainly from cell-free supernatants(CFS). The antibacterial activity of CFS kept within p H3.0~4.5,and lowered by treating with neutral protease,pepsin and papain,but was not sensitive to α-amylase.The antagonistic activity remained by thermal treatment from 40 ℃ to 121 ℃ for 30 min. The inhibitory substance of LAB produced was preliminary determined as type II non- glycosylation bacteriocin. Strain LY- 19 was identified as Lactobacillus sakei according to physiological and biochemical characteristics and 16 S r DNA sequence analysis.