Staphylokinase (Sak), produced by lysogenic strains of Staphylococcus aureus, can convert plasminogen into its proteolytic form, plasmin, and thus is widely used to dissolve pathological clots in clinical applications. In the present paper, we report a novel approach to produce r-Sak (recombinant Sak) using an engineered Escherichia coli expression system. The expression plasmid was constructed by placing the Sak gene into the expression vector pET32(a), resulting in the expression of 35% fusion protein. Subsequently, a rapid and simple chromatographic procedure was developed for the large-scale purification of therapeutic-grade r-Sak from E. coli, which includes Ni(2+)-affinity chromatography, ultrafiltration and Q-Sepharose Fast Flow chromatography. This method led to the production of highly pure r-Sak (>99%), according to SDS/PAGE and HPLC analysis.