Objective To develop an analytical method for determination of seven trace neonicotinoid pesticides in milk by using ultra-high performance liquid chromatography-high resolution mass spectrometry. Methods The samples were extracted, enriched and cleaned up using acetonitrile-water based on cold-induced liquid-liquid extraction (LLE) and dispersive solid-phase extraction (DSPE) method. The chromatographic separation of analytes was conducted on an HSS T3 column (2.1 mm×100 mm, 1.8 μm) using acetonitrile-water system as the mobile phase with gradient elution. The analytes were detected by high resolution mass spectrometry using target single ion monitoring mode and the internal standard method was used as the quantitative analysis. Results Seven neonicotinoid pesticides showed a good linearity in a certain concentration range with relative coefficient of R²>0.999. Limits of detection (LODs) were in the range of 3-7 ng/L, and limits of quantitation (LOQs) were between 8 and 20 ng/L. At the spiked levels of 20,100, 1 000 ng/L, the average recoveries of analytes in milk were between 89.4%-105.7%, with the intra-day precision (RSD_r) of 0.8%-6.4% and the inter-day precision (RSDR) less than 11.0%. Conclusion Cold-induced LLE and DSPE method allows one-step sample preparation. This method demonstrates a rapid, simple and low-cost pretreatment with high sensitivity and good reproducibility, which is suitable for trace analysis of neonicotinoid pesticides in milk.