A gas chromatography-mass spectrometry (GC-MS) method was developed for determining β-sitosterol in milk by optimizing important sample pretreatment parameters such as saponification temperature, extraction solvent, extraction number, shaking method during extraction and rotary evaporation temperature. The feasibility of the developed method was examined. This method exhibited a wide linear range and could meet the requirements for routine quantitative analysis of β-sitosterol in milk samples with high precision, good repeatability, accurate results and high reliability.