AbstractThe luminescence performance of fluorescent reagents plays a crucial role in fluorescence analysis. Therefore, in this study, a novel bi‐ligand Zn‐based metal–organic framework, Au nanoparticle (NP) fluorescent material was synthesized using a hydrothermal method with Zn as the metal source. Simultaneously, a DNA aptamer was introduced as a molecular recognition element to develop a Zn‐based MOF@Au NPs/DNA aptamer fluorescent probe for the ultra‐trace detection of thiamethoxam residues in agricultural products. The probe captured different concentrations of the target molecule, thiamethoxam, through the DNA aptamer, causing a conformational change in the DNA aptamer and bursting the fluorescence of the probe, therefore establishing a fluorometric method for thiamethoxam detection. This method is highly sensitive due to the excellent luminescence properties of the Zn‐based MOF@Au NPs, and the DNA aptamer can specifically recognize thiamethoxam, offering high selectivity. The linear range of the method was 2.5–6000 × 10−11 mol L−1, with a detection limit of 8.33 × 10−12 mol L−1. This method was applied to the determination of actual samples, such as bananas, and the spiked recovery rate was found to be in the range 84.05–109.07%. Overall, the proposed probe has high sensitivity, high selectivity, and easy operation for the detection of thiamethoxam residues in actual samples.