Avian influenza virus (AIV) remains a persistent threat to global poultry health and production, necessitating the development of novel and effective vaccination strategies. This study evaluated the immunogenicity and protective efficacy of a newly developed bovine adenovirus-vector-based avian influenza virus (BAdV-AIV) vaccine expressing the hemagglutinin (HA) gene of H5N1 and the nucleoprotein (NP) gene of H7N9 AIV subtypes (BAdV - (H5N1)HA+(H7N9)NP). In our study, the candidate vaccine was administered via three mucosal routes: oral (OR), intranasal (IN), or intraocular (IO), using three different doses (10?, 10?, and 10? pfu/bird) in a prime-boost vaccination regimen in broiler chickens. Immunological analyses revealed that high-dose vaccination, administered via the IO route induced a substantial upregulation of virus-specific systemic IgY, hemagglutination inhibition (HI) antibody titers, mucosal sIgA antibody responses in bile, trachea and intestines, and activation of various T-cell subsets (helper T cells, cytotoxic T cells, and γδ T lymphocytes). The quantitative PCR (qPCR) assay failed to detect a difference in the challenge H5N2 AIV genomic equivalent load in the vaccinated groups. A qualitative analysis for live challenge virus detection was performed by inoculating the tracheal swab fluid into embryonated chicken eggs and harvested allantoic fluid was tested by hemagglutination assay. Our data showed a lower percentage of tracheal samples yielding detectable live virus in the high-dose BAdV-(H5N1)HA+H7N9)NP AIV vaccine group, particularly following IO administration, compared with the other vaccination routes. Overall, the BAdV-(H5N1)HA+ (H7N9)NP vaccine elicited AIV-specific humoral, mucosal, and cellular immune responses and was associated with a reduced percentage of virus-isolation-positive tracheal samples, particularly following IO administration. These findings support the need for further evaluation of this vaccine platform for avian influenza control in poultry.