Abstract: To develop a detection method for Streptococcus agalaciate in tilapia, a double-antibody sandwich ELISA (DAS-ELISA) was established using the SIP monoclonal antibody (McAb) as the capture antibody and the HRPlabelled rabbit polyclonal antibodies against SIP as the detecting antibody.Two hybridoma strains, 1C9 and 6G5, that secreted antibody against SIP of S.agalaciatein tilapia, were prepared and characterized.The titers of these two monoclonal antibodies could reach 1∶105 and 1∶104, and their isotypes were IgM.Indirect ELISA assay indicated that McAb 1C9 had a specific and sensitivity detection for S.agalaciate.Polyclonal antibodies against SIP were developed and conjugated with HRP.The titers of them were 1∶105and 1∶104.The optimal concentration of HRP-labelled antibody was 1∶400.The ELISA had no cross-reaction with S.iniae, S.suis or S.pluranimalium, and the minimum amount of S.agalaciate could be detected by this DAS-ELISA was 0.85×106 CFU·mL-1.The results indicated that the ELISA method was quick, sensitive, reproducible and applicable for the detection of S.agalaciatein tilapia.