Abstract: Immunoglobulin of red spotted grouper (Epinephelus akaara), purified by saturation ammonium sulfate precipitation follow with Sephrose-4B chromatography, was used as antigen to immunize BALB/c mice. After cell fusion and screening the antibody secreting cell, two hybridoma cell lines which secreting monoclonal antibodies (Mab) against E. akaara Ig had been established. In isotyping analysis, the results showed that both Mabs were IgG1. ELISA results showed that titer of ascites was 106 and sensitivity of the Mabs to purified E. akaara Ig was as low as 62ng. Further experiments proved that Mab-2D3 only specifically bound to the Ig of E. akaara, while Mab-8D6 not only able to recognize the Ig of E. akaara but also recognizes serum Ig of some marine fishes such as Cuvier et Valenciennes, Lateolabrax japonicus, Pseudosciaena crocea. Mab-8D6 also had low level cross reaction with the serum Ig of Cyprinus carpio and Anguilla anguilla. Western-blotting revealed that both Mabs can recognized molecular weight 790 kD Ig of E. akaara under non-denatured and non-reduced conditions, while neither of the Mabs can recognized the heavy chain and light chain of E. akaara Ig under denatured conditions. All the results demonstrated that Mab-2D3 and Mab-8D6 against different epitopes, Mab-2D3 have stronger specificity and recognize unique epitopes of E. akaara Ig, while cross reaction result of Mab-8D6 suggested that E. akaara have closely evolution relation on Ig with above marine fishes.