Despite the availability of a vaccine, pertussis is still a worldwide health problem. Outer membrane vesicles (OMVs) in gram-negative bacteria can stimulate the immune system due to several outer membrane proteins and are very good candidates in vaccine development. OMVs obtained from Bordetella pertussis contain several antigens, which are considered immunogenic, and could make them a potential candidate for vaccine production. The current study aimed to compare the current OMV extraction method (with ultracentrifuge) and a modified extraction method (without ultracentrifuge) and to evaluate the physicochemical properties as well as the expression of their main virulence factors. Vaccinal strain BP134 grown on Bordet Gengo agar were inoculated in Modified Stainer-Scholte medium for mass cultivation. OMVs were prepared using two different methods. They were then stained and examined with a transmission electron microscope. Protein contents were measured by the Bradford method, and then the protein profile was evaluated by SDS-PAGE. The presence of immunogenic antigens was detected by Western blotting. The size and shape of the OMVs obtained from the modified method without the use of ultracentrifuge were similar to the current method and had a size between 40 and 200 nm. The total protein yields of the OMV isolated using the current and modified methods were 800 and 600 µg/ml, respectively. Evaluating the protein profile of extracted OMVs showed the presence of different proteins. Finally, the presence of PTX, PRN, and FHA was observed in OMVs extracted from both methods. Comparison of the two OMV extraction methods showed that the obtained vesicles have a suitable and similar shape and size as well as the expression of three important pathogenic factors as immunogens. Despite the relatively low reduction in protein yield as the modified method does not require ultracentrifuge, this extraction method can be used as a suitable alternative for extracting the outer membrane vesicles from B. pertussis, especially in developing countries. It should be noted that further experiments including immunogenicity determination of OMVs obtained as vaccine candidates in animal models are required.