Schmallenberg virus (SBV), discovered in continental Europe in late 2011, causes mild clinical signs in adult ruminants, including diarrhoea and reduced milk yield. However, fetal infection can lead to severe malformation in newborn offspring. To develop improved reagents for SBV serology, a high-level yeast expression system was employed to produce recombinant SBV nucleocapsid (N) protein. Recombinant SBV N protein was investigated as an antigen in SBV-specific IgG enzyme immunoassay and used for generation of monoclonal antibodies (MAbs). Yeast-expressed SBV N protein was reactive with anti-SBV IgG-positive cow serum specimens collected from different farms of Lithuania. After immunization of mice with recombinant SBV N protein, four MAbs were generated. The MAbs raised against recombinant SBV N protein reacted with native viral nucleocapsids in SBV-infected BHK cells by immunofluorescence assay. The reactivity of recombinant N protein with SBV-positive cow serum specimens and the ability of the MAbs to recognize virus-infected cells confirm the antigenic similarity between yeast-expressed SBV N protein and native viral nucleocapsids. Our study demonstrates that yeast expression system is suitable for high-level production of recombinant SBV N protein and provides the first evidence on the presence of SBV-specific antibodies in cow serum specimens collected in Lithuania.