Background and Objectives: There are many pertussis outbreaks which is mainly due to the reduction in the immunity of acellular pertussis (aP) vaccines. Therefore, there is a crucial necessity to develop a new generation of pertussis vaccine. Preceding researches have shown that Bordetella pertussis outer membrane vesicles (OMVs) have appropriate specifications, making them a suitable vaccine candidate against pertussis. Materials and Methods: The OMVs were separated by a new serial ultra centrifugation technique. Transmission electron microscopy (TEM) examination, SDS-PAGE, Western blotting and ELISA assay were used to characterize the OMVs. Results: TEM studies showed the size of the extracted OMVs at 40–200 nm. The presence of pertussis toxin, filamentous hemagglutinin, and pertactin was verified using Western blot and ELISA assay. Conclusion: The presented technique is a simple and effective way to obtain OMVs from Bordetella pertussis. So it can be utilized as an appropriate procedure in the development of an OMV-based vaccine against pertussis.