In this work, we present the in silico design of a new aptamer (named Apt917) capable of interacting with E. coli and its evaluation as a recognition element in electrochemical biosensors. A search and bioinformatic analysis of various aptamers reported in the literature was carried out. Parameters such as low dissociation constant and short sequences were considered to design and model new hybrid sequences. The designed Apt917 has a minimum free energy and high percent to frequency, making it promising compared to the initial ones. Apt917 was synthesized and immobilized on gold nanoparticles modified screen printed electrodes. The evaluation of the obtained biosensor shows a promising detection of E. coli O157:H7 with limits of detection and quantification of 8 and 27 CFU/mL, respectively.