The aim of this study was to evaluate the usefulness of gene recombination technique using the pET-30 Ek/LIC expression vector for production a 36 kDa released protein called YopD and evaluate of this purified protein as antigen in serodiagnosis of yersiniosis. Protein YopD of Y. enterocolitica was expressing in Escherichia coli BL21 (DE3) using the pET-30 Ek/LIC expression vector. Purification of the expressed enzyme from suspensions of E. coli cells treated with Bug Buster Protein/Extraction Reagent was accomplished by immobilised metal (Ni2+) affinity column chromatography (His-trap). The IgM, IgG and IgA class antibodies to YopD were measured in 100 serum samples collected from patients suspected for yersiniosis and 100 blood donors. The obtained results were compared to the results of ELISA with released proteins isolated from the culture of Y. enterocolitica supernatant under calcium deficient conditions and commercial ELISA with recombinant released proteins. A very high (94.0-100.0%) specificity and good sensitivity (55.2-80.4%) were displayed by the ELISA with YopD in relation to other two ELISA. The results of our study showed that recombinant YopD protein purified by chromatography of bio-affinity may be used in serodiagnosis of yersiniosis as a high specific antigen free of Yersinia lipopolysaccharides.