Abstract: The aim of this study was to prepare the specific monoclonal antibody (McAb) against LPS of B．melitensis, and to develop a sandwich ELISA for detecting B．melitensis. The LPS from B. melitensis strain 16M was purified by using hot phenol extraction method, and identified by SDSPAGE. Six to eightweekold female BALB/c mice were immunized with inactivated thalli of B. melitensis (strain 16M) in Freund’s complete adjuvant and LPS in Freund’s incomplete adjuvant successively. Three hybridoma cell lines secreting McAb against LPS were obtained and named as strains 5H3，6B8 and 3H7, respectively. The ELISA titers of cell cultures supernatant and ascites fluids of all three hybridoma cell lines ranged from 1∶1 000 to 1∶5 000, and 1∶10 000 to 1∶160 000, respectively. Immunoglobulin subclass tests differentiated corresponding mcAbs from the three hybridoma cell lines as IgG3 (3H7) and IgM (5H3 and 6B8). The specificity assay showed that mAbs from 6B8, 5H3 and 3H7 had noncrossreactivity with antigens from Colibacillus O157, Salmonella pullorum, Flexneri Shigella and LPS of Gallibacterium anatis, and only reacted with the inactivated thalli of B. melitensis (strain 16M). RoseBengal plate and tube agglutination assays indicated that the acquired mAbs could react with standard antigen of B．melitensis, which further demonstrated that the mAbs from 6B8, 5H3 and 3H7 had strong specificity. A sandwich ELISA for detecting B．melitensis was developed by using the mcAbs produced from hybridoma cell lines. Simulative samples and clinical samples were tested with the developed sandwich ELISA. Results showed that this ELISA had very high accuracy. Specific hybridoma cell lines secreting monoclonal antibodies against LPS Antigen of B. Melitensis were successfully developed, and provide a basis for establishing rapid diagnostic method of Brucella Melitensis.