The invention discloses a latex agglutination kit for detecting streptococcus agalactiae antibodies of dairy cows as well as a preparation method and an application of the kit. The kit comprises latex agglutination particles, positive control serum, negative control serum, a sample diluent, a glass slide and a graduated pipette. The kit is used for detecting whether the dairy cows suffer from mastitis, is simple, convenient and fast in method, has strong specificity and high sensitivity and is suitable for being used for detection by grass-roots veterinarians; and instruments and equipments are not needed.