Staphylococcus aureus is a pathogen responsible for purulent infection in humans and animals. To prevent disease of the nasopharynx, it is significant to prepare specific egg-yolk antibodies for determination establishment. In this paper, egg yolk antibody against staphylococcus aureus was prepared by immunization of laying hens with straphylococcus aureus as antigen. Egg yolk immunoglobulin (IgY) was isolated and purified from the egg yolks by polyethylene glycol 6000 and ammonia sulfate solution method. The titer and purify of IgY was evaluated by enzyme-linked immunosorbent assay (ELISA) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) respectively. Bacteriostatic effect was assayed with bacteriostatic test by plate method. Cross-reactivities with other bacterias were investigated by indirect ELISA. The specificity of IgY and antigen combination was assayed with agar-gel immunodiffusion method. The purity of IgY is 95.2 % and the titer is 1:5120. The IgY is with high specificity of streptococcus aureus. It can bind to staphylococcus aureus antigen well. The results indicate it provide a new approach to determine and prevent staphylococcus aureus.