The increasing prevalence of lupin allergy as a consequence to the functional characteristics of a growing number of sweet lupin-derived foods consumption makes the imperious necessity to develop analytical tools for the detection of allergen proteins in foodstuffs. The current study developed a new highly specific, sensitive and accurate ELISA method to detect, identify and quantify the lupin main allergen β-conglutin (Lup an 1) protein in natural and processed food. The implementation of accurate standards made with recombinant conglutin β1, and an anti-Lup an 1 antibody made from a synthetic peptide commonly shared among β-conglutin isoforms from sweet lupin species was able to detect up to 8.1250 ± 0.1701 ng (0.0406 ± 0.0009 ppm) of Lup an 1. This identified even lupin traces present in food samples which might elicit allergic reactions in sensitized consumers, such as β-conglutin proteins detection and quantification in processed (roasted, fermented, boiled, cooked, pickled, toasted, pasteurized) food, while avoiding cross-reactivity (false positive) with other legumes as peanut, chickpea, lentils, faba bean, and cereals. This study demonstrated that this new ELISA method constitutes a highly sensitive and reliable molecular tool able to detect, identify and quantify Lup an 1. This contributes to a more efficient management of allergens by the food industry, the regulatory agencies and clinicians, thus helping to keep the health safety of the consumers.