Nowadays, food allergy is a very important health issue, causing adverse reactions of the immune system when exposed to different allergens present in food. Because of this, the development of point-of-use devices using miniaturized, user-friendly, and low-cost instrumentation has become of outstanding importance. According to this, electrochemical aptasensors have been demonstrated as useful tools to quantify a broad variety of targets. In this work, we develop a simple methodology for the determination of β-lactoglobulin (β-LG) in food samples using a folding-based electrochemical aptasensor built on poly-L-lysine modified graphite screen-printed electrodes (GSPEs) and an anti-β-lactoglobulin aptamer tagged with methylene blue (MB). This aptamer changes its conformation when the sample contains β-LG, and due to this, the spacing between MB and the electrode surface (and therefore the electron transfer efficiency) also changes. The response of this biosensor was linear for concentrations of β-LG within the range 0.1-10 ng·mL −1 , with a limit of detection of 0.09 ng·mL −1 . The biosensor was satisfactorily employed for the determination of spiked β-LG in real food samples.incidence of the most popular food allergies throughout Europe [13] revealed that allergy to cow's milk is, nowadays, the most frequent food allergy, especially in early-age children, implying a percentage of around 6% in the youngest population [14,15]. Immunoglobulin E (IgE) normally mediates cow's milk allergy, and the symptoms usually occur within 2 h after milk intake [16]. In whey from cow's milk, one of the most important proteins considered to be an allergen is β-lactoglobulin (β-LG), which is classified within the lipocalins, a group of binding proteins with small ligands [17]. Its quaternary structure, identified in 1997 using X-Ray diffraction [18], is dependent on the pH. For example, at pH values between 5.2 and 7.0, it is a dimer, and its molecular weight (MW) is around 37 kDa. However, at pH values over 8.0, it exists as a monomer, and its MW is approximately 18 kDa [19]. This protein has a great potential as milk allergen because it is remarkably stable and it is not present in human milk [20], and, apart from that, it is regularly used as an additive in lots of food products [21].The development of analytical approaches used to detect and quantify food allergens has become increasingly significant [22] because labeling in food is crucial for the consumers, in order to prevent major health problems. The analysis of allergens in food samples [23] is usually carried out by immunoassay-based approaches, such as enzyme-linked immunosorbent assays (ELISAs) [11,14,24] or lateral-flow immunoassays (LFIAs) [25,26], as well as DNA-based methods, which detect the genes encoding for the proteins using amplifying methods, for instance, the polymerase chain reaction (PCR) [27,28]. Proteomic tests have been also used for the determination of allergens [29]. Although these methods are very sensitive, they are time-consuming and the instrumenta...