Gluten contamination is a constant risk for people with celiac disease (CD), necessitating the development of specific and ultra-sensitive methods for detecting Gliadin. Here, we report the first use of a screen-printed gold electrode (SPGE) modified with a novel nanocomposite of gold nanourchin/molybdenum disulfide/titanium dioxide (AuNU/MoS2/TiO2) in Nafion to detect Gliadin using electrochemiluminescence (ECL). The nanocomposite enhanced ECL intensity by 5.6 times compared with the bare SPGE. The immunosensor had a linear detection range of 0.005–500 pg/mL, with a low limit of detection (LOD) of 0.005 pg/mL, the lowest among other Gliadin immunosensors. The immunosensor exhibited high selectivity, reproducibility, and stability, and demonstrated excellent recoveries (98.0%–104.9%) of Gliadin from real samples. The proposed immunosensor has the potential to provide a rapid and accurate gluten detection tool for the food industry, and to help protect CD patients against gluten exposure.