Despite numerous advancements in gluten detection, a substantial need remains for innovative, cost-effective, in situ methods that can be employed without complex analytical instruments. Addressing this demand, this study introduces a pioneering label-free colorimetric biosensor for the in situ detection of gliadin, a major component of gluten, which is a prevalent trigger of food allergies. Our novel approach employs the strategic coating of gold nanoparticles (AuNP) with gliadin-specific aptamers. In the absence of gliadin, these aptamers stably disperse AuNP, preventing their aggregation. However, upon the introduction of gliadin and in the presence of sodium chloride, AuNP aggregate, yielding a measurable colorimetric signal that facilitates the precise quantification of gliadin. Under rigorously optimized conditions, this AuNP/aptamer-based colorimetric biosensor demonstrated exceptional sensitivity and selectivity, with a detection limit of 32.1 ng mL-1 and a linear response range of 0-300 ng mL-1. Critically, the sensor maintained reliable performance when applied to real-world food samples, including gluten-free bread, cookies, and pasta. Due to its simplicity, selectivity, speed, and cost-effectiveness, this assay represents a significant advancement over current gluten detection methods. Moreover, the developed AuNP/aptamer-based colorimetric biosensor design holds promising potential for adaptation to detect other food allergens or protein toxins through selective aptamer modifications.