Enterokinase (EK) is an amino protease existing in duodenum of mammal and is involved in digestion. It consists of a disulfide-linked 82–140 kDa heavy chain which anchors enterokinase in the intestinal brush border membrane and a 35–62 kDa light chain which contains the catalytic subunit. Additionally, both of the chains are derived from a single precursor that is cleaved by a trypsin-like protease. EK can specially recognize the amino acid sequence DDDDK, and digest the peptide bond after the lysine residue. rEK was report to be more effective than nature EK in cleaving recombinant proteins. Furthermore, the light chain possesses the whole enzyme activity of EK. rBoEK has the highest activity than EK of other species and is used wildly in biochemical applications.