Staphylococcal enterotoxin B (SEB) excreted by Staphylococcus aureus is a common cause of food poisoning. Moreover, SEB is one of the harmful or hazard agents used as biological weapons in bioterrorism or biological warfare. Thus, the creation of nontoxic mutant SEB with immunological reactivity for production of monoclonal antibodies and for further development of detection kit of SEB is of great interest. In the present study, the mutant SEB in the form of nontoxicity was produced by replacing four histidinesof the wild type SEB with tyrosines at positions 12, 32, 105, and 121 by Mega-primer method, respectively. The mutant was cloned and ligated into pET22b+ expression vector to establish pET22b+/mtSEB recombinant plasmid. The cell clones carrying recombinant DNA was selected by colony - PCR and digestion with EcoRl and HindIII restriction enzymes. The results of this study showed that mutant seb gene containing four site mutations (SEB H12Y/H32Y/H105Y/H121Y) was created by wild-type SEB gene (length of 534 nucleotides) in S. aureus isolated from Vietnam and successful designing pET22b+/mtSEB recombinant vector. The mutant SEB obtained will be used as biological materials for later studies in order to develop quick detection kit of SEB, such as rapid lateral flow strip kits or biosensors.