A method for separating diastereomers of pristane. A pristane sample is prepared, and then injected into a chromatographic instrument equipped with a chiral chromatographic column, where a stationary phase of the chiral chromatographic column has a preset pore size. The pristane diastereomers in the pristane sample are separated by the chiral chromatographic column, and the components produced by the separation of the pristane diastereomers sequentially enter a mass spectrometer for detection and analysis.