Currently, the detection of pathogens such as Escherichia coli through instrumental alternatives with fast response and excellent sensitivity and selectivity are being studied. Biosensors are systems consisting of nanomaterials and biomolecules that exhibit remarkable properties such as simplicity, portable, affordable, user‑friendly, and deliverable to end‑users. For this, in this work we report for the first time, to our knowledge, the bioinformatic design of a new peptide based on TIR protein, a receptor of Intimin membrane protein which is characteristic of E. coli. This peptide (named PEPTIR‑1.0) was used as recognition element in a biosensor based on AuNPs‑modified screen‑printed electrodes for the detection of E. coli. The morphological and electrochemical characteristics of the biosensor obtained were studied. Results show that the biosensor can detect the bacteria with limits of detection and quantification of 2 and 6 CFU/mL, respectively. Moreover, the selectivity of the system is statistically significant towards the detection of the pathogen in the presence of other microorganisms such as P. aeruginosa and S. aureus. This makes this new PEPTIR‑1.0 based biosensor can be used in the rapid, sensitive, and selective detection of E. coli in aqueous matrices.