Objective：To construct the expression vector of human staphylokinase （SAK） gene，express SAK protein in E.coli，purify recombinant SAK protein and to verify its bioactivity. Methods：On the basis of bioinformatics，gene sequence was analyzed and op－timized，SAK plasmid was constructed by gene synthesize methods and the protein was expressed by B834 strain. The recombinant protein was purified by ion exchange coloum （DEAE） and size exclusion chromatography（Superdex 75）. Soluble fibrin plate method was used to characterize its fibrinolytic activity. Results：The recombinant SAK was highly expressed in soluble supernatant by E coli system. The recombinant protein with 95% purity was obtained by using of DEAE and Superdex 75.Experiments in vitro revealed that the fibrinolytic activity of SAK was approximately equal to that of urokinase. Conclusion:The recombinant SAK with high fibrinolytic activity can be highly expressed in E coli system by utilizing sequence optimizing and gene synthesize.