Bluetongue (BT) is an arbovirus transmitted disease by bites of the genus Culicoides and infects wild and domestic ruminants particularly in sheep. As an important outer shell protein which defines BTV serotypes, VP2 has been shown to be an ideal target antigen for identification of different BTV serotypes. In order to prepare a monoclonal antibody (mAb) against the VP2 protein of BTV-4, the corresponding encoding gene L2 was divided into three segments and then cloned into pET-28a (+) and pMAL-c5X vectors to generate recombinant plasmids, which were expressed in Escherichia coli BL21 (DE3) as histidine (His)-tagged (His-4A/4B/4C) and maltose binding protein (MBP)-tagged (MBP-4A/4B/4C) fusion proteins. After affinity purification of His-4A/4B/4C with Ni-NTA agarose and MBP-4A/4B/4C with amylose resin, His-4A/4B/4C were used to immunize BALE/mice and MBP-4A/4B/4C were used to screen for mAb-secreting hybridomas. Five hybridoma cell lines stably secreting mAbs against different VP2 segments were obtained, in which 4A-1G7 and 4B-1B6 could recognize BTV-4 and also cross-react with other BTV serotypes. With the joint action of the two mAbs, BTV-4 and BTV-20 infection would be distinguished from other BTV serotypes. The successful preparation of recombinant VP2 segments and mAbs provides valuable materials that can be used in serological diagnosis of BTV-4.