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A method using polypyrrole-coated Fe₃O₄ (Fe₃O₄@PPy composites) based extraction coupled with high performance liquid chromatography was developed for adsorption and detection of trace vitamin D₃ (VD₃) in aquatic products. The fabricated Fe₃O₄@PPy composites were characterized by scanning electron microscopy, transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and thermogravimetric analysis. Fe₃O₄@PPy composites showed efficient adsorption of VD₃ at pH 9.0 and 25 °C with a dose of 25 mg per 10 mL of sample solution and an adsorption time of 11 min. Methanol was selected as the desorption solvent to recover VD₃ from Fe₃O₄@PPy composites after 3 min of static treatment. Fe₃O₄@PPy composites can be used for VD₃ adsorption at least two times. The developed method showed a good linearity for VD₃ determination in the range of 0.1–10 μg/mL with a correlation coefficient of 0.9989. The limits of detection and quantification were 10 ng/mL and 33 ng/mL, respectively. The recovery of VD₃ in a spiking test was 97.72% with a relative standard deviation value of 1.78%. The content of VD₃ in nine aquatic products was determined with this method. Our results show that Fe₃O₄@PPy composites provide a convenient method for the adsorption and determination of VD₃ from the complex matrix of aquatic products.