Pyridaben, a broad-spectrum pyridazinone acaricide that is widely used in agricultural production, can induce neurotoxicity and reproductive abnormalities, and is highly toxic to aquatic organisms. In this study, a pyridaben hapten was synthesized and used to prepare monoclonal antibodies (mAbs), among which 6E3G8D7 showed the highest sensitivity in indirect competitive enzyme-linked immunosorbent assay, with a 50% inhibitory concentration (IC50) of 3.49 ng mL−1. The mAb, 6E3G8D7, was further applied to a gold nanoparticle-based colorimetric lateral flow immunoassay (CLFIA) for pyridaben detection, according to the signal intensity ratio of the test line to the control line, which showed a visual limit of detection of 5 ng mL−1. The CLFIA also showed high specificity and achieved excellent accuracy in different matrices. In addition, the amounts of pyridaben in blind samples detected by the CLFIA, were consistent with high-performance liquid chromatography. Therefore, the developed CLFIA is considered a promising, reliable, and portable method for pyridaben on-site detection in agro-products and environmental samples.