The H9N2 influenza A viruses are endemic in some countries for many years and had caused economic losses in poultry industries. Infection with this virus decreases egg production and increases chicken mortality. In human health, cases of this zoonotic agent are being reported and some cases showed fatal infection. The H9N2 is highly contagious, and transmission can occur from chicken to chicken, chicken to humans through the air, or sometimes from humans to humans. H9N2 belongs to alpha influenza virus genus of the family Orthomyxoviridae. It has a minus sense RNA genome of eight segments. This fact makes the virus to easily re-assort, which might yield a new virus strain, and causes an outbreak in unimmunized humans or chickens. In reducing losses due to H9N2 infection, rapid diagnosis is an option to prevent further infections. Office International des Epizooties (OIE) suggested some methods for the detection of H9N2 infection including the immunofluorescence method that is commonly known as the direct fluorescent antibody (DFA) assay. This method is a combination of immunology and bio-imaging. This DFA assay is rapid and sensitive, but specificity is influenced by antibody production methods. Therefore, the aim of this review was to describe various antibody production methods, which might be used in developing DFA assay to diagnose H9N2 infection in humans or chickens, and we discussed avian influenza H9N2 virus, immunogens, various H9N2 antibody production, and principle of DFA assay.