Streptokinase is a very well known novel fibrinolytic protein. It is produced by several species of Streptococci and possesses therapeutic importance. Streptokinase can be dissolves a blood clot by the activation of plasminogen to plasmin. Soil is an excellent source for isolation of enzymes. In the present study a total of 50 soil samples were collected from nearby ENT hospital, 23 isolates were found as streptococci which were isolated on glucose azide medium and demonstrated for β-haemolysis on blood agar. Among them 11 Streptococci strains showed β-haemolysis and subjected to caseinolysis assay and blood clot dissolving assay as well as morphological and biochemical characterization based on Bergey’s criteria. Highest streptokinase activity showed S9 isolates were subjected for the large scale production of streptokinase. The optimum temperature and pH of Streptokinase is 27-37°C and 9 respectively. The molecular weight of Streptokinase was obtained 47 kDa. The thrombolytic potential of this particular isolate indicated that it could extract a promising streptokinase with potent activity also it may be utilized for large scale production of streptokinase.