Three glycoproteins of infectious laryngotracheitis virus (ILTV), gC, gE, and gp60, were expressed in Escherichia coli as fusion proteins with a 6-histidine tag at their amino termini. The proteins expressed, designated as r-gC, r-gp60, and r-gE, all retain their antigenicity, as revealed by Western blot with chicken antiserum against ILTV. However, only r-gp60 and r-gE, but not r-gC, were found to be soluble. The soluble r-gp60 and r-gE were purified by a nickel column and then used as the enzyme-linked immunosorbent assay (ELISA) antigen for detecting ILTV-specific antibodies. The diagnostic potential of r-gE and r-gp60 ELISA was assessed with the use of sera prepared from vaccinated or unvaccinated chickens of either specific-pathogen-free (SPF) or field origins. The result shows that r-gp60 and r-gE ELISA could discriminate vaccinated SPF chickens from unvaccinated ones 2 wk postvaccination. Moreover, r-gp60 and r-gE ELISA could also discriminate vaccinated field flocks from unvaccinated ones. This result indicates that r-gp60 and r-gE might serve as an alternative ELISA antigen for detecting ILTV-specific antibodies. Moreover, r-gp60 or r-gE ELISA might play an important role in the eradication of infectious laryngotracheitis (ILT) in the future when the gp60- or gE-deleted marker vaccine of ILT is available.