A solid-phase extraction (SPE) method based on conductive polypyrrole (PPy) nanofibers which were fabricated by electrospinning and in situ polymerization was developed. PPy nanofibers-mediated SPE followed by high performance liquid chromatography (HPLC) was used for the determination of three synthetic estrogens, namely diethylstilbestrol (DES), dienestrol (DIS), or hexestrol (HS) in milk sample. Extraction conditions including extraction nanofibers, donor solution pH and salt concentration were optimized. The target compounds were extracted from a 0.5 mL aqueous sample at pH 5.0 through PPy fibers, and then were eluted with 0.1 mL methanol. After extraction, the eluant was directly injected into an HPLC system for detection. Under the optimized extraction conditions, a large enrichment factor was achieved for three estrogens. The limit of detection (LOD) at a signal to noise ratio (S/N) of 3 ranged from 0.02 to 0.05 µg mL-1 for the estrogens in milk sample.