Acetochlor is an endocrine disruptor. The acetochlor residue is strongly lipophilic and can be enriched into products during the manufacturing process. In this study, we found that dimethyl-β-cyclodextrin (DM-β-CD) solution could decrease the apparent oil/water partition coefficient (Koil-w) of acetochlor and increase the sensitivity of fluorescence lateral flow immunoassay (LFIA) for acetochlor simultaneously. Based on this, a simple LFIA method for the determination of acetochlor and alachlor residues in vegetable oil was established. The detection process only involves vortex mixing of an oil sample and dimethyl-β-cyclodextrin solution in a 1 : 3 (V/V) ratio, loading the water phase onto the immunoassay strips and reading the results. Under optimized conditions, the LOD for acetochlor in oil was 3.53 ng g-1, and the working range was 12.03-2000.00 ng g-1. The recoveries of spiked samples ranged from 91.69% ± 1.12% to 112.23% ± 2.20%. Meanwhile, the cross reactivity for alachlor was 108.22%, while for other investigated acetochlor analogues it was less than 1%, and the recoveries of alachlor were from 92.90% ± 8.03% to 113.53% ± 3.40%, which indicate that this method can detect acetochlor and alachlor simultaneously. Compared with the traditional detection method, the pre-treatment process of the proposed method is "green" and simple, and can be applied to the on-site rapid detection of acetochlor and alachlor in vegetable oil and can provide inspiration for the detection of other lipophilic pollutants.