Objective To prepare the monoclonal antibodies (mAb) against hemagglutinin of H4 subtype avian influenza virus (AIV), and develop a sandwich ELISA for the detection of H4 subtype AIV. Methods The BALB/c mice were immunized with inactive H4 subtype AIV. A mAb against H4 subtype AIV, designated as 6G4, was obtained by cell fusion, hemagglutination inhibition (HI) screening and subcloing. Immuofluorescence cytochemistry and Western blotting were used to detect the reactivity of 6G4 with H4 subtype AIV, and the specificity, broad spectrum and stability of 6G4 were characterized by HI assay. Subclass of 6G4 was determined by kit. With chicken polyclonal antibody against H4 subtype AIV as coated antibody, 6G4 mAb as capture antibody and HRP-labeled goat anti-mouse IgG as the enzyme-labeled antibody, a sandwich ELISA for the detection of H4 subtype AIV was established by optimization of the reaction conditions and serial verification. Results 6G4 belonged to IgG1 subclass, and the light chain belonged to κ. It could secrete antibody stably and had good reactivity, specificity, broad spectrum and stability. ELISA based on 6G4 was specific, sensitive, accurate and suitable for the detection of a large number of samples. Conclusion We successfully achieved the anti-H4 subtype AIV mAb, and developed the sandwich ELISA for the detection of H4 subtype AIV.