Abstract: A strain of lactic acid bacteria S-4, identified as Lactobacillus paracasei S-4 according to the nucleotide sequence of its 16S rDNA, was used to conduct experiments to observe its antagonistic effect on four strains of vibrio. This study compared the antibacterial effect of supernatant with different culture durations and dilution ratios on vibrio and explored the competition of L. paracasei S-4 in co-cultivation with vibrio in vitro. The results showed that the supernatant had no inhibitory activity at 16 h of fermentation but showed inhibitory activity to 4 strains of vibrio after 20 h culture. And 36 h supernatant was the best harvest time for inhibiting V. harveyi, V. vulnificus and V. parahaemolyticus, when the inhibition percentage reached more than 95.42% and there was no significant decrease of inhibitory effect after 3-fold dilution. But to V. alginolyticus, 72 h supernatant was the best harvest time and showed a 95.72% inhibition ratio, and 6-fold dilution of it exhibited no significant difference. L. paracasei S-4 in co-cultivation with vibrio started to reduce the 4 strains of vibrio at 4-6 h, its inhibitory activity appearing 12 h ahead of supernatant, indicating that L. paracasei S-4 was able to inhibit the growth of vibrio at the beginning of culture. Biochemical assay showed that the inhibitory compounds were insensitive to catalase, pepsase and snailase, and ultrafiltration and cold ethanol precipitation could not eliminate their inhibitory activity. But the inhibitory activity disappeared completely when supernatant pH was adjusted to neutral, indicating that organic acid may play an important role in bacteriostatic activity. The results of this study showed that L. paracasei can strongly inhibit 4 strains of vibrio and has potential value of application in aquaculture.