Akabane virus (AKAV) is a Culicoides-borne Orthobunyavirus that is teratogenic to the fetus of cattle and small ruminant species. To develop an effective diagnostic assay for the detection of AKAV, we generated and characterized one AKAV N-reactive monoclonal antibody (mAb) which was designated as 2D3. Western blot and indirect immunofluorescence assays indicated that 2D3 could react with both recombinant N protein and AKAV isolate TJ2016. The linear epitope recognized by mAb 2D3 was located at amino acids 168-182 of AKAV N protein. Then, mAb 2D3 was applied to establish a double antibody sandwich ELISA (DAS-ELISA), which was able to detect both the purified AKAV N protein (with detection limit of 6.25 ng/ml ) and AKAV-infected cell culture supernatant (with detection limit of 250 TCID ₅₀/ml). Taken together, the successfully prepared mAb and the preliminarily established DAS-ELISA provide valuable materials and a promising method for the serological diagnosis of AKAV.