Comprehensive innovative teaching experiment on analysis of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) in water by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was designed according to the latest research results. Solid-phase extraction (SPE) was used to enrich PFOA and PFOS in water, and then PFOA and PFOS were eluted, concentrated and transferred to sample vials for analysis by UPLC-MS/MS. This experiment could not only comprehensively examine students’ experimental skills and scientific literacy, but also systematically train students’ professional knowledge to understand environmental problems by combining with new research on analysis technology of emerging pollutants. Finally, the comprehensive quality of students would be enhanced.