Abstract: Objective: The aims of this study were to optimize an efficient combination of lactic acid bacteria (LAB) with strong antagonistic activity against Vibrio harveyi from the marine fish intestine.Methods: LAB with no cross antagonism were obtained using the method of Oxford cup agar diffusion. Complex LAB with strong synergy inhibition activity against V. harveyi was optimized by orthogonal experiment.Results: Lactic acid bacteria were obtained from the marine fish intestine, which had strong inhibition activity against V.harveyi and had no cross antagonism.The bacterial suspension of strain YP4-5, YP1-6 and DY4-2combined with the ratio of 2 ∶ 3 ∶ 3 reached the maximum zone of inhibitory diameter and reached 25.87 mm at 24 h, which increased 27.06%, 37.10% and 36.73% than that strain YP4-5, YP1-6, DY4-2 respectively.It indicated that the three strains LAB had the synergetic effects.Conclusion: Lb.plantarum DY4-2 and Lb. sake YP4-5, Lb. sake YP1-6 had synergetic effects.against V.harveyi, which combined with the ratio of 2 ∶ 3 ∶ 3 had strong antagonistic activity against V. harveyi. Therefore, the complex LAB can be used as the composite microbial agent for the control of V.harveyi in aquaculture.