Cloning of staphylokinase gene carried out in E.coli JM 109 (DE3). recombinant pSP72 was used to transform competent E.coli JM 109 (DE3) as a new and safe host for different genes and different organisms. Then positive transformants of E.coli (containing recombinant pSP72) was selected on plasma agar medium containing 50 g/ml of ampicillin as a selectable marker. The degree of staphylokinase gene expression in the new host (E.coli) was asymbtotic to the degree of expression of the same gene in the original host (S.aureus). Optimum conditions for staphylokinase gene expression in genetically engineered E.coli was studied under two growth factors. Results of optimization showed that the degree of gene expression in new host was increased when transformant E.coli JM 109 (DE3) was grown in Luria Bertani agar containing IPTG (as inducer for gene expression) in a concentration of 100 g / ml and incubated at 37 C for 24 hours. Effect of incubation temperature on staphylokinase activity produced by genetically engineered E.coli JM 109 (DE3) transformant was studied in range of temperature. Results showed that the optimum temperature for staphylokinase activity was 37°C, at this temperature the diameter of zone of hydrolysis was 30mm, while the enzyme activity was decreased above and under this temperature.