In this study, we synthesized silica-coated magnetic nanoparticles functionalized with second-generation polyamidoamine dendrimer (Fe3O4@SiO2@PAMAM-G2). The Fe3O4@SiO2@PAMAM-G2 nanocomposite was used as a sorbent for the magnetic solid-phase extraction (MSPE) of estradiol valerate from biological samples followed by HPLC-UV assay. The characterization of the nanocomposite was performed by Fourier transform infrared spectroscopy (FT-IR), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), thermogravimetric analysis (TGA), and X-ray diffraction (XRD). Under the optimum extraction conditions, the linear dynamic range was obtained in the range of 0.03–100 ng mL−1 and the limit of detection (LOD) was 0.01 ng mL−1. The relative standard deviations (RSDs %) were in the range of 5.1–6.1% and absolute extraction recovery (ER%) was 89.3%. Finally, the estradiol valerate successfully was extracted and was determined in the human plasma samples.

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