Abstract: In order to establish a sensitive and rapid detection method of the residues of thiamethoxam in cucumber, Brassica chinensis and water samples, the chemiluminescence enzyme immunoassay (CLEIA) of thiamethoxam residues was studied. A CLEIA method was developed by optimizing the working concentration of antigen and antibody, the type of blocker, methanol content, Na⁺ concentration and pH value in the analyzing system. Under the optimal conditions, the linear range of the method was 0.125-12.5 ng/mL, the limit of quantification (LOQ) was 0.125 ng/mL, and the IC₅₀ was 1.01 ng/mL. The cross-reaction rates of the seven analogues of thiamethoxam were all lower than 0.3%, which indicated that the CLEIA method had good specificity. The recovery study in cucumber, Brassica chinensis and pond water showed that the recovery range was 86%-108%, and the relative standard deviation (RSD) was 2.6%-9.1%, which was consistent with the analysis results of ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Using the established method, the residues of thiamethoxam in market samples of cucumber, Brassica chinensis and pond water samples was detected, and the results showed that the residues of thiamethoxam in the three kinds of samples were lower than the detection limit. This study showed that the established CLEIA method was highly sensitive, environmental friendly, the sample preparation was simple, and it can be used in the detection of thiamethoxam residues in cucumber, Brassica chinensis and water samples.