Abstract: Tomato spotted wilt disease caused by tomato spotted wilt orthotospovirus (TSWV) has resulted seriously yield and quality losses in vegetables and flowers in China. In this study, nucleocapsid protein N gene and membrane polysaccharide protein Gn gene of TSWV were cloned into prokaryotic expression vectors, respectively. The plasmids were transformed into Rosetta(DE3) cells for expression of N or Gn proteins, which were used as immunogens to immunize New Zealand white rabbits, then the polyclonal antibodies against N and Gn proteins were successfully prepared. Western blot analysis showed that the polyclonal antibody against N protein could bind to the N protein specifically. Indirect ELISA showed that the titer of antiserum against N protein was about 1:12 800. However, the specificity of antibody against Gn protein was poor, and the titer was not detected. The polyclonal antibody against N protein has been prepared successfully, and it can be a useful reagent for disease diagnosis in field, the research of virus localization in insect body and the interaction of virus and insect vector.