The Staphylococcal enterotoxins (SEs) are a major cause of food poisoning worldwide. In particular, Staphylococcal enterotoxin B (SEB) had been considered as an important agent in biological weapons. Therefore, development of cost-effective, rapid. and sensitive kit for the detection of SEB is of great interest. In this study, the authors conducted expression and purification of mutant SEB (mtSEB) coded by correponding gene of 534 nucleotides containing mutations H12Y, H32Y, H105Y and H121Y to generate recombinant SEB with non or lower toxicity which could be used as biological materials for the production of specific antibodies against superantigen in the development of rapid detection kit of SEB food poisoning in further studies. The .obtaine4 results showed that the recombinant mtSEB534 protein of approximately 22 kDa was expressed in E. coli strain BL21 (DE3) at high level at 37°C, 0.5 mM IPTG in 5 hours. Also, in this study. the recombinant mtSEB534 protein was purified successfully by nickel resin column affinity with the purity reached 95 percent, this is ready for immunization test.