Pine wilt disease is currently the most deadly forest disease in the world and is known to be caused by infection of Bursaphelenchus xylophilus. Until now, no method has been developed to confirm the pine tres infected with pine wood nematodes. In order to develop a method to diagnose pine wood nematode infection, we produced a monoclonal antibody against Expansin B3, which was found to be secreted by pine wood nematodes. ELISA assay using various monoclonal antibody confirmed that the pine trees infected with the rewarming can be detected. Further studies using our antibodies may help to develop a diagnostic method that can quickly confirm infection of pine wood nematodes in the field.