For efficacious DNA vaccine development against infectious bronchitis virus (IBV), the immunogenicity of a multivalent DNA vaccine was evaluated. Three expression plasmids each targeting spike protein (S1), nucleocapsid protein (N), and membrane protein (M) of IBV were prepared. Chickens were immunized with either individual plasmids (monovalent) or with a combination of all plasmids (multivalent). Immunization with the multivalent DNA vaccine induced synergistic augmentation of humoral and cellular responses in comparison with the individual vaccines, and provided up to 85% immune protection. Thus the multivalent DNA vaccine represents an innovative approach for enhancing DNA vaccine potency, and has potential clinical application for vaccination against IBV.