A gel permeation chromatography purification (GPC)-gas chromatography-mass spectrometry (GC/MS) method was developed for the determination of 6 steroid hormone residues (androsterone (A), dehydroepiandrosterone (DHEA), testosterone (T), progesterone (P), estrone (E1) and β-estradiol (β-E)) in butter samples. The butter samples were extracted by oscillation with ethylacetate/cyclohexane (1:1, V/V). The extract was purified and degreased by GPC column. The GPC concentrate was evaporated to dryness under a stream of nitrogen. Derivatization of the residue was carried out with pyridine and aceticanhydride. Finally, the derivative was separated using a GC column (HP-5MS, 30 m×0.25 mm, 0.25 μm), and the qualitative and quantitative analysis was conducted under the model of selective ion monitor (SIM). Steroid hormone residues were quantified using external standard method, which had good linearity in the concentration range of 10-500 μg/kg with correlation coefficiency larger than 0.999. The quantification limit was 4-10 μg/kg. At the spike levels of 10 μg/kg, 50 μg/kg and 200 μg/kg, the recoveries of hormones were in the range of 77.2-112% and the relative standard deviation (RSD) was from 4.2% to 10%. The above results showed that the method was accurate, reliable and suitable for the determination of 6 hormone residues in butter samples.