An acetonitrile (ACN)-salt based aqueous two-phase system was developed and applied to the extraction of steroid hormones in milk. The extraction of hormones was assisted by ultrasonication after adding ACN and sodium chloride and then centrifugation was used to effect the phase separation. The extraction and purification were performed in one step and no further clean-up step is needed. The hormones were determined using high-performance liquid chromatography. The experimental parameters, including type and amount of salt, pH value of the sample solution, extraction temperature and time, and volume of ACN, were optimized. The limits of detection for steroid hormones ranged from 0.90 to 1.52 μg L⁻¹. When the present method was applied to the analysis of milk samples, the recoveries of analytes ranged from 87.3% to 105.8% and relative standard deviations were lower than 7.73%. In one sample, 6.26 μg L⁻¹ of progesterone was detectable.