With the rapid development of aquaculture industry, bacterial diseases in fish are breaking out frequently.Antibiotics are the main methods to prevent and control the bacterial diseasesof fish, but this has been bringing some problems, such as bacterial resistance, drug residues, food safety and environmental pollution.Vaccines to control fish disease outbreaks are the most effective way.Currently, the use of natural polymer materials for carrier preparation of oral microsphere vaccine has become a hot topic.Oral microsphere vaccine can not only prevent gastrointestinal environmental damage to the vaccine, but also achieve all the growth stages of immunization of fish, and reduce the immune costs.In order to optimize the condition of preparation in microsphere vaccine.Pathogenic bacteria Edwardsiella ictaluri were killed by formalin and sodium alginate was used, and then the oral microsphere vaccine of E.ictaluri was prepared by emulsification ionic crosslinking method.Single factors including sodium alginate concentration, water-oil ratio, emulsifier concentration and emulsification time were investigated separately.On the basis of single-factor experiments, the relationships among main preparation conditions were modeled using a 4-factor, 3-level Box-Behnken experimental design.The established model was analyzed by response surface methodology to obtain the optimal preparation conditions.All results showed that the optimal preparation conditions were as follows:sodium alginate concentration was 3.5%, water-oil ratio was 2.4:7.6, emulsifier concentration was 5.4%, and emulsification time was 13 min.Under these conditions, the encapsulation efficiency of microspheres was 96.37%.It matched with the predicted encapsulation efficiency.The microspheres had good form, diameter of particles was homogeneous distribution, diameter(8.88±1.26)μm, span 0.38, remarkable acid resistance, enteric and high security.By response surface methodology, preparation of the oral microsphere vaccine of E.ictaluri.For the development of aquaculture, this vaccine provides a theoretical basis, and the effectiveness of the vaccine will be further studied in subsequent trials.