Objective To establish a double-antibody sandwich ELISA for the rapid detection of shiga toxin typeⅡ ( StxⅡ) in shiga toxin-producing Escherichia coli ( STEC) infection. Methods A pool of murine hybridomas was used to screen out the optimal antibody pair for the establishment of double-anti-body sandwich ELISA. The established ELISA system was used to detect StxⅡin the culture supernatants of 16 clinical strains of STEC. Specificity and sensitivity of the established ELISA system were also evaluated. Results Two antibodies, S2D8 and S2C6, were successfully screened out, based on which the double-anti-body sandwich ELISA was set up. StxⅡand its variants rather than StxⅠwas detected in the culture super-natants of STEC with a lowest detection limit of 4 ng/ml. Its performance was consistent with that of commer-cial colloidal gold test kit, indicating the characteristics of good specificity and sensitivity. Conclusion The S2D8/S2C6-based ELISA laid a foundation for researches which designates the shiga toxin as a potential can-didate on the diagnosis and therapy of STEC infection.