Avian influenza (AI) virus infects both animal and human. Low pathogenic AI virus infections (some H7 and H9 subtypes) have been reported all over the world and pose a potential threat to the poultry industry. Vaccination is the most effective way to prevent virus infection. However, vaccination makes it difficult to differentiate between vaccinated chickens and infected chickens. In order to differentiate vaccinated chickens from naturally infected chickens, we adopted synthetic peptide-based enzyme-linked immunosorbent assay (ELISA) using the peptide sequences from nonstructural protein 1 (NS1) of H9N2. Five synthetic peptides were designed using Protein Variability Sever (http://imed.med.ucm.es/PVS/) and synthesized. NS1-1 ~ NS1-4 peptides failed to detect serum antibodies from both vaccinated and naturally infected chickens. NS1-5 peptide from the C-terminal NS1 protein detected serum antibody from naturally infected chickens but not vaccinated chickens. These results imply that NS1-5 peptide may be a useful tool to differentiate naturally infected chicken from vaccinated chicken as being used in the synthetic peptide-based ELISA.