Antibacterial activity of Fenneropenaeus indicus hemolymph was mediated by Agglutinin (Ag), a protein of molecular weight 181 kDa with two subunits 97 kDa and 84 kDa. Agglutinin was purified by gel filtration chromatography, PAGE, and SDS-PAGE, and polyclonal antibodies (anti-Ag) were raised in rabbits. In a double immunodiffusion test, single precipitin lines were found between the center well containing immune serum and surrounding wells containing purified Ag and crude hemolymph. The IgG fraction was purified from anti-Ag immune serum and used to develop ELISA. The Ag standard curve was linear over a range of 17.8–8,900 ng. The sensitivity of the ELISA was 178 ng/ml. Agglutinin-free hemolymph yielded background optical density values, confirming the specificity of the assay. Serial dilutions of agglutinin-rich hemolymph produced sample titration curves that are parallel to the Ag standard curve. Thus, the ELISA is suitable for quantification of Ag in hemolymph.