The objective of the study was to obtain the gene of bovine enterokinase light chain, which would be used in the cleavage and purification of fusion proteins. The fragment of bovine enterokinase light chain cDNA was obtained by RT-PCR from a sold bovine's duodenal mucosa, then cloned into the pUCmT cloning vector and sequenced. Compared with the sequence deposited in GenBank,the cloned gene sequence is correct. Then the interested gene fragment was inserted into the pET39b expression plasmid. The recombinant vector pET39b-EKL was transformed into E.coli BL21(DE3) and induced by IPTG. It was confirmed that the nucleotide sequence was correct on the conjunction site between the recombinant DNA 5'terminal multi-cloning site and recombinant fragment after the analysis of the nucleotide sequence. SDS-PAGE analysis indicated that target product was about 65 kDa which occupied 28% of the total protein. A pure fusion protein was obtained by nickel chelating chromatogram using His*Binding Resin. After desalting and changing buffer, the crude kinase was incubated at 21 degrees overnight and demonstrated a high autocatalytic cleavage activity. This investigation would be able to lay a foundation for enterokinase activity research and farther application of expression products on a large scale.