Due to its unique insecticidal and acaricidal mechanism of action, and ability to mix with most insecticides and fungicides, diafenthiuron (DIAF) is widely used in the cultivation of fruit and vegetables. However, this insecticide can cause unacceptable harm to organisms, making the detection of DIAF residues in fruit and vegetables crucial. In this study, a novel hapten based on the structure of DIAF was utilized to prepare a monoclonal antibody (mAb) with high specificity and sensitivity. The half maximum inhibitory concentration (IC50) of the anti-DIAF mAb was 20.962 μg/kg as determined by ic-ELISA, and little cross-reactivity with other analogues. Next, a GNP-based lateral flow immunoassay (LFIA) was developed to detect DIAF in cabbages and apples. The optimized LFIA, for cabbage samples, showed a visual limit of detection (vLOD), cut-off value and calculated limit of detection (cLOD) of 0.1 mg/kg, 10 mg/kg and 1.5 μg/kg, respectively, and for apples 0.1 mg/kg, 5 mg/kg and 3.4 μg/kg, respectively. Recovery rates in cabbage and apple were 89.3% -107.2% and 105.3% -112.4%, with a coefficient of variation of 2.76% -5.81% and 2.15%-7.53%, respectively. These results indicated that the established LFIA based on our anti-DIAF mAb was a reliable method for in-situ rapid detection of DIAF in cabbage and apple samples.