Fusing the Myeloma cells SP2 /0 was fused with spleen from Escherichia coli O157∶ H7 immune mice.Three hybridoma cell lines( D3,E7 and B9) secreting anti EHEC O157∶ H7 monoclonal antibody were obtained after screening,wherein the subtype of D3 and B9 belonged to IgG1,the subtype of E7 belonged to IgG2a,and their light chain subtypes all were κ. The cross reaction results showed that these 3 lines of monoclonal antibody didn't respond to the other 15 bacteria strain except the EHEC O157∶ H7. The best pH for monoclonal antibody labeled with Colloidal gold and their binding capacity were as follows: pH7. 5 ~ 8. 0 and 24 μg /mL for D3,pH7. 5 and 12 μg /mL for B,pH7. 5 and 18 μg /mL for E7. The selected best antibody pairs were the optimal combination of gold pad sprayed with colloidal gold-labelledD3 and NC film coated with 1 mg /mL E7 as T line. The sensitivity of test strip for detection of EHEC O157∶ H7 was 2. 1 × 106CFU /mL. The other 25 bacteria strains had no cross-reactivity. The results from bacteria simulation experiments showed that EHEC O157∶ H7 was detectable in commercially available bread,milk and jelly after 200 CFU EHEC O157∶ H7 was added into 25 g /mL each of them and enrichment cultured for 8 h,10 h and10 h. This study showed that the independent preparated antibody has good performance,and the specificity and sensitivity of test trip were similar as those of foreign products,thus it could be widely used in government food-borne pathogen regulatory authorities and food enterprises.