Alkhurma virus (ALKV) is a recently discovered class-4 flavivirus that was responsible for several cases of severe haemorrhagic fever in humans in Saudi Arabia. It has been shown for other flaviviruses that processing of the viral polyprotein is partly due to the virus-encoded NS2B/NS3 trypsin-like serine protease. As the viral proteinase plays a critical role in the virus replication cycle, it represents one of the main targets for antiviral therapy against members of the Flavivirus genus. We report here on the identification of the ALKV NS2B and NS3 domains and the expression and purification of a catalytically active viral protease as a hexahistidine recombinant protein. Its enzymatic properties were characterized in vitro using a para-nitroanilide substrate. This constitutes the first characterization of the proteinase from a class-4 flavivirus. Our results indicate that the association of NS3 with a short segment of NS2B is necessary and sufficient for protease activity. The developed system could help to identify or design inhibitors potentially active as antiviral drugs against ALKV and other pathogenic flaviviruses.