The detection of vitamin D in complex samples is challenging because of the instability of vitamin D in sample preparation. To resolve this problem. packed-fiber solid-phase extraction (PFSPE) has been successfully developed for the pre-treatment of samples containing vitamin D-2 (D-2) and vitamin D-3 (D-3) in dairy products. Based on PFSPE, vitamin D could avoid oxidation because of the shorter extraction time. The adsorbent materials, polystyrene/polypyrrole (PS/PPy) nanofibers, were prepared by electrospinning of PS and in situ polymerization of PPy on the surface of PS nanofibers. The linearity was within the range of 1-2000 ng mL(-1) in dairy solution, and the recoveries were 91.9-109.1% with a relative standard deviation (RSD) < 13.6%. Therefore, the prepared method could be used for the detection of D-2 and D-3 in dairy products.