Nattokinase (NK) is widely used as the thrombolytic agent due to its low cost as compared to several other thrombolytic agents. In the present study rust samples from iron rod was collected and subjected to isolation process in order to screen the NK production. Based on the morphological, biochemical and molecular characterization, the isolate VITSDVM3 was identified as Bacillus cereus which showed highest similarity to B. cereus strain GY-58. The specific activity was determined by casein hydrolysis assay and found to be 954, 1008, 1168, 1201 U/mg for crude, precipitated, dialysed and purified. The NK bearing clot buster activity was determined further by radial caseinolytic activity followed by fibrin degradation assay which showed 10 and 15 mm respectively. The maximum clot lysis activity was found to be 97.43 %. The stability of the purified enzyme was analysed by various parameters including pH, temperature, surfactants, metal ions and organic solvents. High Performance Liquid Chromatography analysis was performed in order to indentify the retention time. Our preliminary data thus indicated the fibrinolytic effects of NK, and suggesting its potential value as a therapeutic agent and the need for additional studies and clinical trials. © 2016, The National Academy of Sciences, India.