Influenza virus causes acute respiratory disease in pigs and is of concern for its potential public health significance. Many subtypes of influenza virus have been isolated from pigs, and the virus continues to evolve in swine populations. Current antibody assays have limited antigenic recognition, and accurate, broad-spectrum, high through-put screening tests are needed to detect infections in swine herds and to aid in the implementation of control measures. In the current study, a commercial blocking enzyme-linked immunosorbent assay (ELISA) developed for the detection of Influenza A virus nucleoprotein antibodies in avian species was evaluated for the detection of anti-influenza serum antibodies in swine. Serum samples used to evaluate the test were archived samples from influenza research conducted at the U.S. Department of Agriculture-Agricultural Research Service-National Animal Disease Center and included samples from influenza-inoculated pigs (H1N1, H1N2, H2N3, and H3N2), contact-infected pigs, vaccinated pigs, and negative controls. Based on samples of known status (n = 453), a receiver operating characteristic (ROC) curve analysis of the ELISA results estimated the optimized diagnostic sensitivity and specificity at 96.6% (95% confidence interval [CI]: 92.3, 98.9) and 99.3% (95% CI: 97.6, 99.9), respectively. By using the cutoff established in the ROC analysis, the assay was evaluated in pigs infected with 2 isolates of the 2009 pandemic H1N1 virus. Overall, the assay showed excellent diagnostic performance against the range of influenza subtypes investigated and could serve as a useful screening assay for swine.