The present research work is designed to extract the Staphylokinase from Staphylococcus sp. isolated from milk samples. Mannitol salt agar and Blood agar was used to isolate the Staphylococcal isolates. Casein hydrolytic assay and heated plasma agar assay was performed to screen the Staphylokinase producing Staphylococcus sp. Satoh's media was used as the production media for the enzyme. Partial purification and molecular weight of the enzyme was determined by ammonium sulphate precipitation and SDS PAGE analysis. Enzyme activity was confirmed using Holmstrom method. Performing the modified Holmstrom method it was observed that staphylokinase extracted fromStaphylococcus sp.showed thrombolytic activity at concentrations of 80-100 μl. After precipitation of the enzyme, maximum activity was determined at concentrations as low as 10 μl. Molecular weight of the extracted staphylokinase was determined as 14.5 kDa. Thus this research was successfully carried out to find an economical and safer clot buster agent compared to chemical agents such as EDTA or heparin.