Abstract: A polythiophene (PTh) in-tube solid-phase microextraction (PTh/IT-SPME) column was prepared, and a method for detecting residues of 24 pesticides in water was established by combining high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Pesticide residues in 7 actual samples were detected, and the adsorption mechanism of the polythiophene coating for different pesticides was explored. After conducting samples using a PTh/IT-SPME column, elution was performed with a solution of 0.5% formic acid in acetonitrile, followed by detection using HPLC-MS/MS in multiple reaction monitoring (MRM) mode. 24 pesticides exhibited good linearity within the range of 1-200 μg/L and all had coefficients of determination (R²) greater than 0.999. The limits of quantitation (LOQ) of all pesticides were 0.2 μg/L. The spiked recoveries of 24 pesticides at concentrations of 0.2 and 2.0 μg/L were in the range of 62%-97%, with relative standard deviation (RSD) of 1.4%-7.1%. Two pesticides were detected in the actual samples, with concentrations all complying with the maximum residue limits specified in relevant national standards. The experimental results of the adsorption of pesticides on PTh/IT SPME columns show that the adsorption behavior of the PTh coating for pesticides was dominated by π-π interactions between the PTh and pesticide molecules, which was also influenced by the polarity, planar structure, charge distribution, and the steric hindrance of molecules. This method features short pretreatment time, minimal organic solvent usage, and excellent extraction performance, making it suitable for rapid detection of multiple pesticide residues in water.