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The use of affinity peptide displaying on the surface of M13 phage has emerged as a promising alternative to antibody-based detection methods owing to its diverse structure, mass production, easy purification, and genetic manipulation, as well as excellent stability. In this study, we aimed to identify highly selective and sensitive peptide-displayed M13 phage for thrombin detection using phage display technology. Phage display was conducted using two different phage libraries (linear and cyclic forms of peptides), which displayed affinity peptides capable of binding specifically to thrombin. Throughout the biopanning process, two phage clones that displayed both peptides (WGQSFWGYQHYS and CWKLLTPWC) were selected. After the characterization of their binding abilities using enzyme-linked immunosorbent assay, whole phage particles were covalently attached to a gold electrode using MUA-EDC/NHS coupling chemistry. The thrombin detection mechanism was based on signal inhibition, and the results revealed that the specific interactions between the phage-tethered gold electrode and thrombin are critical factors affecting the decrease in the peak current of the sensor. In addition, under optimal conditions, the fabricated phage-based electrochemical sensors were capable of highly sensitive and specific thrombin detection with a good binding affinity (Kd = 17 nM for 12mer 3–17 and 14 nM for C7C 3–5) and limit of detection (4 nM for 12mer 3–17 and 10 nM for C7C 3–5), resulting in good reproducibility, even in artificial human plasma samples. This study may provide an alternative and more efficient method of detecting thrombin with desirable sensitivity, selectivity, and feasibility for the diagnosis and prognosis of coagulation disorder-related diseases. [ABSTRACT FROM AUTHOR]