Introduction: In spite of high vaccination coverage, whooping cough (pertussis) is still a worldwide health problem. The main reason for pertussis outbreak is waning immunity of safer acellular vaccines which have replaced the more reactogenic cellular vaccines. A new generation of pertussis vaccines that is potent and safe is desperately needed to control the disease. Previous studies have indicated that outer membrane vesicles (OMVs) obtained from Bordetella pertussis have desirable characteristics which make them a good candidate for application as pertussis vaccine. They contain surface immunogens in a native structure, are self-adjuvant and are easily uptaken by the antigen presenting cells. Methods: B. pertussis Tohama strain was cultured at 35°C in Stainer-Scholte broth. The OMVs were isolated by a new sequential ultracentrifugation method. The extracted OMVs were characterized by electron microscopy, SDS-PAGE and ELISA assays. Results: The existence of pertussis toxin, filamentous haemagglutinin and a 69-kDa antigen in B. pertussis OMVs was verified using an ELISA assay. Electron microscopy showed the size of these OMV’s at 40-200 nm. The ELISA results indicated that the OMVs extracted using this protocol contain major immunogens. Conclusion: We report for the first time a simple protocol for the efficient extraction of B. pertussis OMVs. This protocol can be used in the process of making new generations of B. pertussis vaccines.