The hemagglutination inhibition (HI) test is the current gold standard for detecting antibodies to avian influenza virus (AIV). Enzyme-linked immunosorbent assays (ELISAs) have been explored for use in poultry and certain wild bird species because of high efficiency and lower cost. This study compared a commercial ELISA for detection of AIV subtype H5 antibodies with HI test of 572 serum samples from zoo birds. There was no significant difference between the results of the two tests when statistically compared by a McNemar χ² test (P = 0.86) and assessment of κ (κ = 0.87). With a specificity of 94.2% (95% confidence interval [CI], 0.92–0.97), a sensitivity of 93.9% (95% CI, 0.91–0.97), and an excellent correlation between the two tests, this ELISA can be recommended as an alternative to the HI test for preliminary screening of zoo bird sera for antibodies to AIV subtype H5.