Microbiological contamination caused by foodborne diseases has become a major public health problem of the world. A novel electrochemical immunosensor assay (EIA) for high sensitive and specific detection of Escherichia coli O157:H7 was developed. A new nanocomposites with multiwalled carbon nanotubes (MWCNTs) embedded inmetalorganic coordination polymers (MOCPs) was successfully prepared as highly efficient matrices of capturing antibody immobilization for sensitive electrochemical biosensing. In the presence of target E. coli O157:H7, horse radish peroxidase (HRP)labeled antibody was captured on the electrode surface to form a sandwichtype system via the specific identification. As a result, E. coli O157:H7 detection was realized by outputting a redox current from electroreduction of hydrogen peroxide reaction catalyzed by HRP. In the assay, the combination of the unique properties of MWCNTs and MOCPs can not only accelerate electron transfer on the electrode interface, but also provide an excellent scaffold for the conjugation of capture antibody. Meanwhile, adopting the MWCNTs-MOCPs materials significantly improved the target capturing efficiency and enhanced the sensitivity of the biosensor. The results revealed that the calibration plot obtained for E. coli O157:H7 was approximately linear from 67 cfu/mL to 6.7×106 cfu/mL with the limit of detection of 40cfu/mL. In addition, the biosensor was successfully applied to quantitative assay of E. coli O157:H7 in synthetic sample (milk). Hence, the developed electrochemical based immunosensor might provide a useful and practical tool for E. coli O157:H7 determination and related food safety analysis and clinical diagnosis.