Bacterial diseases continue to be a major economic factor for commercial catfish farming. Correct diagnosis of diseases is, therefore, important for effective treatment measures and to prevent production losses. The present study aimed at fractionating and characterizing the serum globulin proteins of pangas catfish, Pangasius pangasius by salt fractionation and SDS-PAGE analysis. The protein profile of fractionated serum globulin proteins of P. pangasius revealed a total of 12 bands, viz., 136.64, 120.58, 95.87, 79.92, 72.67, 63.04, 45.99, 43.32, 38.65, 34.82, 28.46 and 19.08 kDa. Anti-pangas serum globulin (ASG) horseradish peroxidase (HRPO) immunoconjugate (ASGHRPO immunoconjugate) was prepared following sodium periodate procedure and raising immune serum against it in rabbit. The sero-reactivity of ASG-HRPO immunoconjugate was assessed by dot-ELISA and direct ELISA. In dot-ELISA, development of intense colour spot was noted only when ASG-HRPO immunoconjugate was used either at neat or 1:10 dilution. Similarly, the direct ELISA results showed low sero-reactivity with a dilution titer value <200 against pangas serum globulin protein fraction as antigen. Dot-ELISA was also performed to detect the presence of specific antibodies against Aeromonas hydrophila and Edwardsiella tarda in P. pangasius separately by experimental inoculation. Development of intense colour spot was observed in both cases, which in turn indicated the specificity of ASG-HRPO immunoconjugate in detecting the anti- A. hydrophila and anti- E. tarda antibodies in P. pangasius serum. The development of colour spot upon the addition of sensitized fish immune serum and incubation suggested that the ASG-HRPO immunoconjugate can be used for the seromonitoring of specific antibodies and serodiagnosis of diseases caused by E. tarda and A. hydrophila.