Influenza A virus continue to cause widespread morbidity and mortality. The unprecedented spread of highly pathogenic avian influenza virus subtype H5N1 in Egypt is threatening poultry and public health systems. Effective diagnosis and control management are needed to control the disease. To this end, polyclonal antibodies (PAbs) were developed against the H5N1 avian influenza virus (AIV) and used in an enzyme-linked immunosorbent assay (ELISA) to detect the H5 viral antigen. A group of rabbits were immunized with H5N1 vaccine to obtain PAbs as the detector antibody after conjugation with horse radish peroxidase and fluorescein isothiocyanate (FITC). The conjugated PAbs proved to be specific for the detection of AIV in field specimens, and results were confirmed using reference antisera obtained from Veterinary Lab in Weybridge. Specimens collected from different governorates of Egypt and tested positive for AIV by haemagglutination test were used to evaluate the produced PAbs. The detection limit of ELISA using the prepared peroxidase conjugated PAbs was 1:100,000, while the limit using fluorescein conjugated PAbs was 1:10,000. Extracts from pharyngeal-tracheal mucus of apparently healthy chickens mixed with H5 AIVs also yielded positive signals in ELISA. Such data suggest that these PAbs are useful in the surveillance and diagnosis of AIV in birds in Egypt.