Abstract The increasing incidence of infectious outbreaks from contaminated food and water supply continues imposing a global burden for food safety, creating a market demand for on‐site, disposable, easy‐to‐use, and cost‐efficient devices. Despite of the rapid growth of biosensors field and the generation of breakthrough technologies, more than 80% of the platforms developed at lab‐scale never will get to meet the market. This work aims to provide a cost‐efficient, reliable, and repeatable approach for the detection of foodborne pathogens in real samples. For the first time an optimized inkjet printing platform is proposed taking advantage of a carefully controlled nanopatterning of novel carboxyl‐functionalized aptameric ink on a nitrocellulose substrate for the highly efficient detection of E. coli O157:H7 (25 colony forming units (CFU) mL −1 in pure culture and 233 CFU mL −1 in ground beef) demonstrating the ability to control the variation within ±1 SD for at least 75% of the data collected even at very low concentrations. From the best of the knowledge this work reports the lowest limit of detection of the state of the art for paper‐based optical detection of E. coli O157:H7, with enough evidence ( p > 0.05) to prove its high specificity at genus, species, strain, and serotype level.