Avian infectious bronchitis (IB) is one of the major respiratory diseases in poultry. At present, attenuated vaccines are the main commercial vaccines, but they have many defects. We aimed to construct a novel multi-epitope DNA vaccine based on avian infectious bronchitis virus (IBV) S1 and N proteins for the prevention of IBV infection. We screened the dominant B and T cell epitopes of target proteins utilizing epitope prediction tools. A new high-immunogenicity epitope peptide segment named QSN was designed and screened by linking peptide. The physicochemical properties of QSN were analyzed by bioinformatics. The recombinant plasmid pEGFP-QSN was obtained by inserting the synthesized QSN gene into the eukaryotic expression vector pEGFP-N1. On the 7th day of age, chicks were immunized by intramuscular injection of the plasmid, and serum specific antibody IgG, cytokines IFN-γ and IL-2, and T lymphocyte subsets were detected after booster immunization. Bioinformatics analysis showed that QSN had high hydrophilicity without transmembrane region and stable structure after binding to receptor. The recombinant eukaryotic vector was successfully constructed. Two weeks after booster immunization, compared with NS group and pEGFP-N1 group, serum IgG level, concentrations of cytokines IFN-γ and IL-2, and proportion of CD4