Objective To effectively and conveniently detect pathogenic bacteria, this study aimed to develop label-free biosensors fabricated affinity peptides that can recognize targeted bacteria strains and enable precise quantitative detections. Results A 12-mer peptide with high binding affinity toward Escherichia coli O157:H7 was discovered by biopanning of phage-displayed peptide library. The peptide modified with glycine residues (G(3)) and one cysteine (C) residue at C-terminal, could self-assemble on gold electrodes, enabling electrochemical impedance spectroscopy (EIS) analysis for quantitative detection of E. coli O157:H7. This method showed a low detection limit of 20 CFU/mL and a liner range from 2 x 10(2) to 2 x 10(6) CFU/mL. Conclusion It appears that, by designing and optimizing the structures of peptides, such a strategy can be greatly promising in developing quick, sensitive and quantitative biosensor of pathogens.