H7N9 subtype avian influenza virus (AIV) is a great threat for poultry industry in China. Serological assays that differentiate infected and vaccinated animals (DIVA) can facilitate H7N9 virus monitoring in vaccinated poultry. A serological method based on a peptide for DIVA diagnosis of H7N9 subtype AIV was previously established. However, complex manufacturing techniques are required for production of chemically-synthesized peptides, casting the affordability and accessibility issues of such assays in poultry farms. Here, to develop an H7N9 DIVA assay suitable for poultry use, a recombinant DIVA peptide fusion protein (DPFP) was expressed in E.coli and its potential as a diagnostic antigen for H7N9 DIVA was assessed. The DPFP composed of the E. coli thioredoxin A, the H7N9 DIVA peptide and His affinity tag was efficiently expressed in a soluble form in E.coli. The DPFP was recognized by H7N9 virus infection serum rather than by H7N9 vaccination serum as determined using immunoblotting. In addition, enzyme-linked immunosorbent assay based on the DPFP was established and optimized, which can well differentiate H7N9 virus infected and vaccinated chickens. OD450 of 0.5 was defined as the cut-off value for H7N9 DIVA diagnosis. Our study indicates that prokaryotic expression system can be used as a promising platform for preparation of diagnostic antigen for discriminating H7N9 virus infected and vaccinated chickens. The method established herein can be employed as an effective and affordable approach for diagnosis and control of H7N9 avian influenza in poultry.