The current study was intended to mitigate the risks posed by Cardiovascular heart diseases(CVD) which is the major cause of mortality and morbidity in the recent years around the world. Staphylococcus produces an enzyme known as Staphylokinase(SAK). In this experiment the SAK producing Staphylococcus sp.,was isolated from the environmental sample (bovine milk) using the selective media (Mannitol salt agar) and further the isolate was allowed to grow on blood agar to check its hemolytic activity.Beta hemolytic colonies were observed on blood agar. The preliminaryscreening of the isolatewere done using Gram Staining, biochemical analysis (catalase and coagulase test) and casein hydrolysis agar assay (for proteolytic activity). The isolate was found to be positive in all preliminary screening tests. Following screening,16S rRNA sequencing was done for species characterization. The Staphylokinasewas produced using the production media(Satoh's media). The enzyme was further extracted and subsequently assayed by Modified Holmstrom method and heated plasma agar assay for thrombolytic and plasmolyticactivity respectively. In Modified Holmstromit was observed that the crude enzyme extracted from the isolate showed thrombolytic activity at concentrations of 100-125µl. Furthermore, the partial purification of the enzyme was done using Ammonium sulphate precipitation.The precipitated protein was then subjected to SDS-PAGE to determine its molecular weight. Also the precipitated protein was then purified using 14 kdaultra filtrationmembrane.As the enzyme was found to be potent in lysing the blood clot, it can be further utilized by the pharmaceutical industries for the production of a clot busting agent which will be useful in treating various cardiac diseases mainly myocardial infarction. © RJPT All right reserved.