Abstract: The study aimed to establish a rapid and simple assay to detect Shigalike toxin IIe (SLTIIe). Monoclonal antibodies (McAbs) against SLTIIe A subunit were produced by using Escherichia coli (E. coli) expressed recombinant SLTIIe A subunit protein as antigen to immunize BALB/c mice. A competitive ELISA for detecting antibodies against SLTIIe was developed on the basis of recombinant protein expressed by E. coli and HRPlabelled McAbs against SLTIIe A subunit. The optimum conditions of the ELISA were developed as following: the concentration of recombinant for coating ELISA plates was 032 μg·mL-1; the best dilution of serum to be tested was 1∶2; the working titer for HRPlabelled McAbs was 1∶3 200; and the inhibition rate above 40% was selected as the positive judging standard. A total of 60 serum samples were detected in parallel by both competitive ELISA and vitro neutralization assay. 33.8% of them were detected as positive by competitive ELISA, while 309% of them were positive by vitro neutralization assay. The coincidence rate of the two assays was 882%. The results showed that the competitive ELISA had the advantages of higher sensitivity, specificity, reproducibility, stability and easy operation. It would be very useful in diagnosis, surveillance of SLTIIe antibodies and epidemiological survey.