In this study, an immunohistochemical (IHC) technique for detecting Newcastle disease (ND) virus (avian avulavirus 1) using paraffin sections of formalin-fixed tissues was developed by production of novel monoclonal antibodies. Mice were immunized with ND virus isolate APMV1/chicken/Japan/Fukuoka-1/2004 (genotype VII), and 14 mouse hybridoma clones producing IgG antibodies against the ND virus were established. Among these antibodies, only three reacted with the viral antigen on the paraffin sections, while one (22C483) exhibited the highest sensitivity in IHC analysis. Results of immunoprecipitation, western blot analysis, and indirect enzyme-linked immunosorbent assay showed that 22C483 reacted with the nucleoprotein of ND virus. Thus, IHC analysis using 22C483, in combination with enzyme digestion treatment for antigen retrieval, can be used for elucidating the pathogenesis of ND.