In analytical chemistry， separation and enrichment steps serve not only as critical stages in the analytical workflow， but also as core technologies that determine the accuracy， reliability， and applicability of analytical methods. However， in conventional experimental instruction， this step is frequently overlooked owing to time constraints and an overemphasis on instrumental techniques， leading to fragmented knowledge structures and hindering the development of integrated analytical thinking that connects theory with practice. To address this issue， this study， based on the “knowledge-ability-practice” three-dimensional integrated teaching objective， innovatively establishes a “positive-negative case-based teaching” model， using the solid-phase microextraction （SPME） of six nitrogen-containing pesticides from environmental water samples as the instructional case. By comparing fibers coated with non-polar polydimethylsiloxane （PDMS） and strongly polar polyacrylate （PA）， and employing high performance liquid chromatography with ultraviolet detection （HPLC-UV）， this study systematically investigates the matching relationship between analyte polarity and coating properties. In the positive case， the PDMS coating demonstrates superior extraction performance for weakly polar pesticides based on the “like dissolves like” principle， with a linear coefficient of determination R²≥0.993 7 and detection limits ranging from 0.019 to 0.17 μg/L. In contrast， in the negative case， the PA coating shows weaker extraction efficiency due to differences in the extraction mechanism， yielding detection limits between 0.066 to 1.069 μg/L. Based on spiked recovery tests conducted on actual river water samples from Lanzhou （recoveries： 81.5% to 117%）， a multi-dimensional evaluation system of “knowledge internalization， competency enhancement， and practical literacy” is established accordingly. Teaching feedback demonstrates that the students show a marked improvement in their understanding of the core principles， and their innovative thinking and problem-solving skills are effectively cultivated. The “contrasting-case-based” pedagogical approach effectively bridges theory and experiment while stimulating students’ investigative thinking. The study provides a replicable and scalable practical model for the reform of analytical chemistry experimental teaching， which holds significant value in cultivating high-caliber talents with rational method selection and problem-solving abilities.