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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.