We present a facile approach for the direct attachment of cross-linked poly(2-thiopheneacetic acid) onto the surface of magnetite nanoparticles with one single carbon layer (Fe 3 O 4 @1C NPs) via self-assembly oxidative polymerization. The functional Fe 3 O 4 @1C NPs combined with poly(2-thiopheneacetic acid) Fe 3 O 4 @1C@ poly(Th) NPs was successfully prepared and used for magnetic solid-phase extraction (MSPE). The influence of the preparation conditions and extraction conditions were investigated in detail. The morphology and size distribution of the monolithic materials were characterized by transmission electron microscopy. Under the optimized experimental conditions, the prepared Fe 3 O 4 @1C@ poly(Th) NPs showed good extraction efficiency for canthin-6-one alkaloids. Furthermore, the results indicated that the binding specificity and selectivity of the Fe 3 O 4 @1C@ poly(Th) NPs might be attributed to electrostatic interactions and pore structure. A MSPE–HPLC method was also developed for quantitative analysis of canthin-6-one alkaloids. The developed method showed good linearity, low limit of detections, low limit of quantifications, and good reproducibility over a wide linear range. To further prove the utility, Fe 3 O 4 @1C@ poly(Th) NPs-MSPE was also used to extract canthin-6-one alkaloids from Eurycoma longifolia stems, with a recovery rate of 87.9–114%. Thus, this method is potentially applicable for the extraction of similar alkaloid compounds from medicinal herb samples.