Japanese encephalitis (JE) is one of the most important viral encephalitis, caused by the Japanese encephalitis virus (JEV). The function of non-structural protein 2B (NS2B) mostly remains unclear. In our study, NS2B of Japanese encephalitis virus (JEV) was expressed in Escherichia coli and purified by dialysis. After fusing mouse myeloma cell line SP2/0 with spleen lymphocytes from NS2B protein immunized mice, three clones of monoclonal antibodies (MAbs), named 1B9, 3E12, and 4E6, were generated. The specificity and sensitivity of MAbs were demonstrated by ELISA, indirect immunofluorescence assay, and Western blot. These MAbs will be useful in further exploration of the functions of NS2B and the pathogenesis of Japanese encephalitis virus.