Avian influenza viruses continue to pose significant zoonotic and pandemic threat globally, with low-pathogenic avian influenza A(H9N2) being of particular concern due to sustained circulation in poultry, adaptability, and repeated human spillover. This study investigated the detection and genetic characterization of influenza viruses at the animal–human interface in Ghana in 2022, using a nationwide cross-sectional One Health approach. Samples were collected from poultry, pigs, the environment, and animal handlers across backyard farms, commercial farms, and live bird markets. Laboratory testing was conducted using real-time RT-PCR, while statistical associations were assessed using chi-square and logistic regression. Whole-genome sequencing and phylogenetic analysis were performed on selected positive samples. Out of 4056 samples, 1516 were poultry samples. A(H9N2) was detected exclusively in poultry, with a prevalence of 5.67%. The Northern belt recorded the highest prevalence. Live bird markets had significantly higher odds of A(H9N2) detection compared with commercial farms (odds ratio: 15.37, p < 0.0001), while backyard farms had lower odds. Environmental samples were negative. Among animal handlers, one case each of A(H3N2) and SARS-CoV-2 was identified. Phylogenetic analysis demonstrated that Ghanaian strains belonged to clade G1 and possessed mammalian-adaptive markers. These findings highlight ongoing circulation in poultry and the need for sustained One Health surveillance.