[Background] Riemerella anatipestifer causes septicemic and exudative diseases of domestic ducks, gooses and turkeys, leading to economically devastating to poultry industries. A total of 21 serotypes of R. anatipestifer have been identified. Moreover, there is poor cross-protection among these serotypes. Lipopolysaccharide is the important component of outer membrane of Gram-negative bacteria, and provides the diversity of bacterial surface antigen determinants. [Objective] To prepare and characterize monoclonal antibodies (McAbs) against lipopolysaccharide of R. anatipestifer serotype 2 strain. [Methods] BALB/c mice were immunized with inactivated R. anatipestifer serotype 2 strain NJ3. Spleen cells from immunized mice were fused with murine myeloma SP2/0 cells. The hybridoma cell line that stably secret McAb against LPS of R. anatipestifer serotype 2 strain, was obtained through clone selection and screening by indirect enzyme linked immunosorbent assay (ELISA). The mouse ascites was prepared and identified by Western blot analysis and ELISA. The specificity of these McAbs was tested by slide agglutination assay and Western blot. [Results] We successfully obtained 2 hybridoma cell lines named 8G5 and 8G10 that could stably produce anti-LPS McAbs. The isotype of both McAbs was identified as IgM with κ light chain. The titers of 8G5 and 8G10 were 1:32 000 and 1:16 000, respectively. Western blot results showed that both McAbs specifically reacted with R. anatipestifer serotype 2 strains but did not react with R. anatipestifer other serotypes strains, avian pathogenic Escherichia coli, Salmonella enterica and Pasteurella multocida strains. [Conclusion] We prepared two specific anti-LPS McAbs that can be used for pathogenic mechanism research and development of rapid detection of R. anatipestifer serotype 2 strains.