Staphylokinase has strong tendency to polymerize, which may hamper its clinical use. On the basis of crystal structure of staphylokinase, the dimer structure was modeled by software GRAMM V1.03 with high-resolution generic docking parameters. Two kinds of binding interfaces were generated, mainly linked by hydrophobic interaction and hydrogen bonds. The computer model of staphylokinase dimer provides a template for the design of improved staphylokinase without tendency to form polymer. A novel variant of staphylokinase (RGD-Sak) was designed based on the model. RGD-Sak gene was constructed by substitution mutagenesis of Lys-109 and Phe-111 with Arg-109 and Asp 111 respectively, confirmed by nucleotide sequencing. The mutant cDNA was ligated with prokaryotic expression vector pLY-4 and transformed into E. Coli JF1125. After temperature induction, over 50% expression level of RGD-Sak was achieved. RGD-Sak was isolated and purified by washing and solubilization of inclusion body, renaturation and ion exchange chromatography. The final product displayed a single band with a corresponding molecular weight of 15.5 kD in non-reducing SDS-PAGE with 95% of purity and 5×10⁴ HU/mg of specific activity. The K_M、K_cat value for the activation of plasminogen with RGD-Sak was 12.40 μmol L^-1 、0.81 s^-1, respectively. RGD-Sak show lower tendency to polymerize after incubated with 0.9% NaCl. This study provided the basis for further reconstruction of Sak by protein engineering.