Abstract: In order to evaluate the optimal processing conditions of egg yolk for the detection of yolk antibody instead of serum antibody in commercial layers which were immunized with Newcastle disease and avian influenza vaccine, in the experiment, 56 healthy commercial layers（Hailan Brown） with egg laying rate of more than 90% were used to obtain serum on 40 days post immunization with Newcastle disease and avian influenza vaccine, and egg yolk was separated from one egg per chicken. A completely randomized experimental design was adopted, and four groups were randomly divided into test groups Ⅰ, Ⅱ, and Ⅲ and the control group. Each group had 56 replicates, each with one serum and one corresponding egg yolk. The control group was tested for serum antibody; the test groups Ⅰ, Ⅱ and Ⅲ were tested for yolk antibody with yolk diluted 1∶1 with normal saline, 1∶3 with normal saline, and 1∶3 with chloroform, respectively. Hemagglutination inhibition（HI） test was used for serum and processed egg yolk antibody detection. The results showed that when detecting Newcastle disease virus and Avian influenza virus H9 antibody in egg yolk, the yolk was diluted 1∶3 with normal saline; when detecting Avian influenza virus H7, Avian influenza virus H5-11 and Avian influenza virus H5-12 antibody in the egg yolk, the yolk was diluted 1∶1 with normal saline. After treatment, there was no significant difference between the yolk antibody titer and the serum antibody titer（P＞0.05）, and the correlation coefficients with the serum antibody titer were 0.837, 0.853, 0.888, 0.888, 0.839, respectively. The results suggested that after the yolk was processed under the suitable conditions, the yolk antibody could be used instead of the serum antibody to evaluate the antibody level of commercial layers.