Preparation of egg yolk immunoglobulin (IgY) against Streptococcus pneumonia is significant to solve the problem of antibiotic resistance and to seek a new therapy of treating, suppressing Streptococcus pneumonia infection. Therefore, we immunized laying hens with antigen of Streptococcus pneumonia and extracted, purified IgY from the yolks of eggs by the acetic acid-sodium acetate buffer dilution, polyethylene glycol 6000 and ammonia sulfate solution method, then the characteristics of IgY were detected. We evaluated the activities of IgY by indirect ELISA, SDS-PAGE electrophoresis, Coomassie brilliant staining blue method, bacteriostatic test and agar gel diffusion test. The purity of IgY was 95.4% and the titer was 1:5120. 8.2 mg IgY could obtained from per milliliter egg yolk. The bacteriostatic test showed that specific IgY against Streptococcus pneumonia could obviously inhibit the growth of Streptococcus pneumonia. The specific combination of IgY and Streptococcus pneumonia could be observed by agar gel diffusion test. In conclusion, IgY against Streptococcus pneumonia obtained from hens immunized by Streptococcus pneumonia could provide a novel alternative approach to treatment and diagnosis of Streptococcus pneumonia infection.