Shigella spp. are one of the most important etiological factors for people who are living in developing countries and travelers to tropical countries. High priority has been given by the World Health Organization to the development of vaccines to control Shigellosis caused by these bacteria. However, information regarding to profile of immunogenic proteins of Shigella is not available now. In the present study, sub-immunoproteomics was applied to screen novel immunogenic proteins which could be reacted with antisera produced by challenge of a whole bacterium. Our results indicated that 13 immunogens were identified, in which seven proteins and six proteins from outer membrane and soluble proteome, respectively. Of the 13 proteins, 12 showed to be novel immunogens. These results suggest that immunoproteomics can greatly improve the chances of identification and result in discovery of novel immunogenic proteins.