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A novel ultrasound-assisted magnetic solid-phase extraction (UA-MSPE) was developed for the separation/preconcentration of trace amounts of pyrethroids (fenpropathrin, fenvalerate, deltamethrin, and bifenthrin) in Paris polyphylla sample using carbon nanotubes based on Fe3O4 magnetic nanoparticles (Fe3O4@CNT MNPs), and high-performance liquid chromatography-UV is described. High recoveries of pyrethroids were obtained at a low MNPs concentration because sonication enhances the contact chances between magnetic nanoparticles and extractable analytes and promotes the extractability of the MSPE process. After the extraction, the adsorbent can be conveniently separated from the sample solution by an external magnet, and the adsorbed analytes were eluted from magnetic Fe3O4@CNT. The main factors influencing the extraction efficiency including the amount of the MNPs, the extraction time, the pH of sample solution, the sonicating time, and the desorption conditions were studied and optimized. Under the optimized experimental conditions, a good linearity was observed in the range of 1-100.0 ng mL−1 for all the analytes, with the correlation coefficients (r) ranging from 0.9962 to 0.9991. The limits of detection of the four pyrethroids are 0.53, 0.26, 0.47, and 0.67 ng mL−1, respectively. The recoveries of the method were in the range between 85.5% and 93.2%. This method is much faster and more effective than traditional SPE methods, and it is promising for the analysis of pyrethroids residues. [ABSTRACT FROM AUTHOR]