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Reliable detection of decreasing amounts of gluten in food is the only way of ensuring the safety of all coeliac patients. Results obtained with the method of choice, immunochemical assays, are not entirely comparable and many of them are sandwich assays that cannot recognize hydrolyzed proteins. In this work, we propose a competitive electrochemical sensor based on a recently described aptamer targeting the gliadin immunodominant peptide 33-mer that triggers the coeliac disease. The sensing layer is built on the surface of a screen-printed carbon electrode (SPCE) by adsorption of streptavidin and subsequent peptide immobilization. A competition between the peptide and gluten proteins from samples for a defined concentration of biotinylated Gli 4 aptamer is established. The aptamer bound to the peptide on the surface is finally measured after enzyme labelling and chronoamperometric detection of an enzymatically obtained electrochemically active product. This method is able to detect as low as 0.113 μg L −1 of gliadin, which corresponds to 380 μg kg −1 of gluten in food, taking all dilutions and conversion factors into consideration, with a reproducibility lower than 11%. The aptasensor was applied to food samples with gluten contents above and below the legislated threshold for gluten-free labelling in the EU, obtaining good agreement with the official R5 immunochemical method. [ABSTRACT FROM AUTHOR]