Interferon-gamma (IFN-gamma), a cytokine produced by sensitized T lymphocytes, is one of the key elements in defining T helper 1 lymphocyte immune responses. Quantitative evaluation of IFN-gamma expression could provide an important analytical tool for measurement of cell-mediated immunity and investigating immune responses to infectious diseases. Method of DNA-designed avian IgY antibodies was used for production of monospecific polyclonal antibodies that allows quantification of the recombinant bovine IFN-gamma protein. IFN-gamma cDNA was subcloned and expressed in mammalian expression plasmid (pcDNA3.1(+)) under the control of the human cytomegalovirus promoter. Chickens were immunized by plasmid DNA, and eggyolk antibodies extracted from eggs were collected after immunization. IgY-specific antibodies were evaluated by an antigen capture enzyme-linked immunosorbent assay (ELISA) using recombinant IFN-gamma. Based on the results, developed bovine IFN-gamma capture ELISA could detect up to 1 ng/ml of IFN-gamma by 64-fold diluted IgY. Monospecific anti-bovine IFN-gamma antibodies generated in chickens are useful for quantifying different concentrations of recombinant bovine IFN-gamma, which is expressed in cell culture.