Staphylokinase is an extracellular protein produced by strains of Staphylococcus aureus which digest fibrin clots. Hence it is widely used as thrombolytic agent due to its low cost as compared to several other thrombolytic agents. The current work has been attempted to characterize the Staphylococcus aureus SPK-7 producing staphylokinase. The strain was subjected to morphological, biochemical and molecular characterization. 16Sr DNA sequencing of the isolate revealed close affiliation with Staphylococcus genera. BLAST search analysis of the sequence showed maximum identity with Staphylococcus aureus CMV201 and Staphylococcus aureus SV4 (99% similarity). Hence the strain SPK-7 was coined as Staphylococcus aureus VITSDVM7. The preliminary screening was done by casein hydrolysis method followed by plasmolytic activity and showed the maximum zone of inhibition of 15 and 17mm respectively. Random mutagenesis was carried out for the enhanced production of staphylokinase by varying time intervals. The purification of staphylokinase from Staphylococcus aureus VITSDVM7 was carried out by ammonium sulphate precipitation, dialysis and gel filtration chromatography resulted in specific activity of 780, 1053, 1105, 1152 U/mg respectively. Eventually there was a gradual increase with corresponding purification fold of 1, 1.4, 1.4, and 1.5. The clot busting activity was found with 50% lysis. Hence the mutated strain produced staphylokinase enzyme which can be considered as a good thrombolytic agent. © 2014, Sphinx Knowledge House. All rights reserved.