Enterokinase is a tool protease widely utilized in the cleavage of recombinant fusion proteins. cDNA encoding the catalytic subunit of Chinese bovine enterokinase (EKL) was amplified by PCR and then fused to the 3' end of prepro secretion signal peptide gene of alpha-mating factor from Saccharomyces cerevisiae to get the alpha-MF signal-EKL-His6 encoding gene by PCR. Then the whole coding sequence was cloned into the integrative plasmid pAO815 under the control of a methanol-inducible promoter and transformed GS115 methylotrophic strain of Pichia pastoris. Secreted expression of recombinant EKL-His6 was attained by methanol induction and its molecular weight is 43 kD. Because of the existence of His6-tag, EKL-His6 was easily purified from P. pastoris fermentation supernatant by using Ni2+ affinity chromatography and the yield is 5.4 mg per liter of fermentation culture. This purified EKL-His6 demonstrates excellent cleavage activity towards fusion protein containing EK cleavage site.