Abstract: Objective: To optimize an efficient complex lactic acid bacteria ( LAB) with strong antagonistic activity against Pseudomonas fluorescens.Methods: The complex LAB strains with synergic and antagonistic effects against P. fluorescens were screened using the method of Oxford cup agar diffusion. The complex LAB were optimized by response surface methodology.Results: The best inhibitory complex of Lactobacillus plantarum DY4-2, Lb. sakei YP4-5 and Lb. sakei LY1-6 were screened from 11 groups of complex LAB against P. fluorescens, and the inhibitory zone diameter reached 25.29 mm, which was higher2.93, 4.80 and 5.35 mm than strain DY4-2, YP4-5 and YP1-6 respectively.Three-factor-three-level experiments designs were developed by Box-Behnken, and the inhibitory zone diameter was used as the responsive values.The bacterial suspension of strain DY4-2, YP4-5 and YP1-6 combined with the ratio of 2∶ 3∶ 1, culture temperature 25 ℃ and culture time 48 h, which the inhibitory zone diameter against P.fluorescens was 25.84 mm in the optimum conditions. Conclusion: The complex LAB can largely increase the synergy between the strain and the inhibitory activity against P. fluorescens, which can provide theoretical basis for the research and development efficient LAB biopreservation control of P.fluorescens in aquatic products spoilage.