Abstract: A novel Flavivirus infection,which mainly caused egg-dropping in ducks and chickens,has spread throughout China since 2010.To carry out the serological surveillance in chickens with minimum stress,an indirect enzyme-linked immuosorbent assay(ELISA) for detecting egg yolk antibody against Flavivirus in layer chickens has been developed by coating plate with chicken-origin Flavivirus FQ-C1 isolate.The optimal working concentration of antigen was 5.7 ng ·μL-1,the working concentration of egg yolk antibody was 1∶160 dilution and that of HRP-labeled goat anti-chicken IgG was 1∶3 200.After a color reaction of 10 minutes with Tetramethylbenzidine(TMB) liquid substrate,a threshold of positive/negative(P/N) ≥ 2.1 was determined as positive standard for the ELISA.The results indicated that the ELISA established by this research provided a high sensitivity and specificity test,which was suitable for the detection of antibodies against Flavivirus in immune laying hens.