Staphylokinase (SAK) is a bacterial kinase produced by certain strains of Staphylococcus spp. It is a 15.5 kDa, consisting of 136 amino acids single chain protein, which activates plasminogen to form plasmin and digest fibrin clots. Staphylokinase (SAK) is a promising thrombolytic agent for the treatment of myocardial infarction. The present research work was carried out to screen the staphylokinase producing Staphylococcus spp. from different samples collected (milk, water and sewage) in and around Vellore district, Tamil Nadu, India. Out of 12 isolates, 4 organisms were producing staphylokinase effectively. The main focus of this work is to isolate and characterise potent producers of staphylokinase producing Staphylococcus spp. from various environmental samples involving non-pathogenic Staphylococcus spp. while the earlier isolation was done using pathogenic samples.