Thrombolytic agents play a major role in the treatment of cardiovascular diseases. Streptokinase is the prominently commercialized thrombolytic drug used for the treatment of cardiovascular diseases. The later studies on staphylokinase (SaK) showed promising results as an alternative fibrinolytic drug. The present study explores the isolation, production and purification of SaK producing Staphylococcus sp. from milk samples. The potent isolate MSA4 of Staphylococcus sp. was selected for production and purification of SaK. The total activity and specific activity of purified staphylokinase was found to be 1266 IU mL-1 and 815.5 IU mg-1, respectively. The partially purified enzyme was lysed the euglobulin clot completely within 18 h of incubation and the purified enzyme showed 79% of blood clot lysis activity. © 2014 Bentham Science Publishers.