Abstract: 【Objective】The vaccine candidate strains of H9N2 subtype avian influenza virus(AIV) with better immunogenicity were screened for new vaccine research and development, which provided technical support for strengthening the prevention and control of H9N2 subtype AIV. 【Method】A total of 36 H9N2 subtype AIV strains isolated in Guangxi from 2000 to 2020 were used as experimental materials. Twelve representative H9N2 subtype AIV strains from different years, different regions and different host sources were selected according to the HA genetic evolution analysis. The cross hemagglutination inhibition test(HI) and antigenicity analysis were carried out with the commercial H9N2 subtype AIV vaccine strain(SS strain) currently used. According to the results of antigenicity analysis, the representative strains with different antigenic differences were selected to prepare oil emulsion inactivated vaccine and tested for crossimmunoprotection with the commercial H9 subtype AIV vaccine strain(SS strain), respectively. 【Result】Among 36strains of H9N2 subtype AIV in Guangxi, 31 strains belonged to Y280-Like(9.4.1), of which 26 strains belonged to branch I, 5 strains belonged to branch II, and the other 5 strains belonged to G1-Like(h9.4.1), which were different from the evolutionary branch of the commonly used vaccine strains in China. The HA titer of 12 H9N2 subtype AIV representative isolates in Guangxi after inactivation was ≥7 log2, and the virus inactivation effect was good, which could be used to prepare inactivated oil emulsion vaccine, and the prepared inactivated oil emulsion vaccine had good stability and safety.The antigen correlation value(R) of each strain was calculated according to the titers of cross-HI antibody. The results showed that there was no obvious difference in antigenicity between A/chicken/Guangxi/C227/2015(H9N2 strain)(referred to as C227 strain) and other strains, and the corresponding R fluctuated between 0.72 and 0.93. The results of vaccine cross-protection test also showed that the immune protection rate of inactivated oil emulsion vaccine prepared by C227 strain was higher than 80% for different strains, and the immune protection effect was better than that of A/chicken/Guangxi/CX/2013(H9N2 strain)(referred to as CX13 strain), which indicated that C227 strain was more suitable as a candidate strain for H9N2 subtype AIV inactivated vaccine. 【Conclusion】A/chicken/Guangxi/C227/2015(H9N2) strain screened based on antigenicity analysis and immunogenicity determination has a good immune protection effect on most H9N2 subtype AIV epidemic strains in Guangxi, and can be used as a candidate vaccine strain to develop a new H9N2subtype AIV vaccine.