The rising prevalence to food allergies in the past two decades, together with the fact that the only existing therapy is avoidance of allergen-containing food next to the implementation of anti-allergic drugs, urges the need for improved performance of current assays to detect potential allergens in food products. Therein, the focus has been on aptamer-based biosensors in recent years. In this paper we report for the first time the selection of aptamers against one of the most important peanut allergens, Ara h 1. Several Ara h 1 DNA aptamers were selected after eight selection rounds using capillary electrophoresis (CE) - SELEX. The selected aptamers specifically recognized Ara h 1 and did not significantly bind other proteins, including another peanut allergen Ara h 2. The dissociation constant of a best performing aptamer was in the nanomolar range as determined independently by three different approaches, being surface plasmon resonance, fluorescence anisotropy, and capillary electrophoresis (353 ± 82 nM, 419 ± 63 nM, and 450 ± 60 nM, respectively). Furthermore, the selected aptamer was used for bio-assay development on a home-built fiber optic surface plasmon resonance (FO-SPR) biosensor platform for detecting Ara h 1 protein in both buffer and food matrix samples demonstrating its real potential for development of novel, more accurate aptamer-based biosensors. In conclusion, the reported aptamer holds a great potential for the detection of Ara h 1 in both the medical field and the food sector due to its high affinity and specificity for the target protein.