Objective: Recombinant technology has crucial impact in therapy development. In microbial environment, pathogenic organism such as staphylococcus aureus produces staphylokinase protein. This protein has major role in thrombolysis. In clinical, non-pathogenic organism well known as Escherichia coli used for recombinant drug synthesis. Methods: In the present study, staphylokinase (SAK) gene is isolated from S. aureus. Sample of pus is collected from wound and infected patients. This S. aureus retrieves from sample. The staphylokinase gene is isolated and injected into a vector such as pET-32 a, b, c (+). This cloned vector transformed into salt induced E. coli strain (DH5α). Results: The mature recombinant Sak protein is expressed in E.coli culture. Further, recombinant staphylokinase is extracted and examined by SDSPAGE and the present of staphylokinase was observed. Conclusion: Recombinant staphylokinase has major crucial role in thrombotic disorders and I will used to design drug against thrombosis without side effect. © 2014 Elsevier B.V., All rights reserved.