Tebuconazole is a widely utilized fungicide for controlling various fungal diseases in crops. However, studies have indicated that tebuconazole may have adverse effects on the human reproductive, digestive, and immune systems. In this study, three monoclonal antibodies (mAbs) targeting tebuconazole were prepared. The ic-ELISA based on mAb 2H1C12 exhibited a half inhibition concentration of 0.23 ng/mL for tebuconazole. The mAb 2H1C12 model constructed with Alphafold 3 was docked with tebuconazole, and the results showed that hydrogen bond and hydrophobic interaction played key roles in recognition Furthermore, a gold nanoparticles-based lateral flow immunoassay was developed using mAb 2H1C12. The LFIA exhibited a detection limit of 2.5 ng/mL in the working buffer and 75 ng/g, 75 ng/g, 35.5 ng/g, and 75 ng/g in apple, cucumber, wheat, and soil samples, respectively. This performance satisfies the detection requirements for tebuconazole in agricultural products as regulated by both China and the European Union. Finally, the qualitative results of LFIA for the blind samples were consistent with the quantitative results of ultra-performance liquid chromatography-tandem mass spectrometry