The BeAn strain of Theiler's murine encephalomyelitis virus (TMEV) causes a demyelinating leukomyelitis in mice, which serves as an important animal model for multiple sclerosis in humans. The present report describes the generation and characterization of a TMEV-specific polyclonal antibody by immunization of rabbits with purified TMEV of the BeAn strain. The specificity of the antibody was confirmed by Western blotting and sequence analysis of the recognized antigen by high resolution mass spectrometry. The presence of TMEV-specific polyclonal antibodies in post-immunization sera was tested on TMEV-infected L-cells (murine lung tumor cell line) using an immunofluorescence assay. Additionally, the rabbit serum enabled virus detection in formalin-fixed and paraffin-embedded TMEV-infected BHK(21) cell pellets and brain tissue of TMEV-infected mice by immunohistochemistry. Immune electron microscopy revealed colloid gold-labeled picornavirus-typical paracrystalline arrays and non-aggregated viral particles of TMEV-infected BHK(21) cells. The present report demonstrates the applicability of the generated marker for investigating TMEV cell tropism and viral spread at a cellular and subcellular level in future studies.