Objectives: To develop a two-dimensional liquid chromatography method to determine the content of vitamin D 3 in cod liver oil preparations., Methods: The samples were prepared by saponification and extraction, and the content of vitamin D 3 was determined by two-dimensional liquid chromatography with dual-pump-single-valve switching dual detectors. The chromatographic column, capture device, and detection wavelength were optimized; the linearity, system suitability, recovery rate, repeatability and sample stability of the method were investigated, and further validated in actual sample determination., Results: A column switching two-dimensional chromatography method was developed. In the first chromatography dimension, an Agilent PoroShell SB-C8 (50 mm×4.6 mm, 2.7 μm) column was used with acetonitrile-water as the mobile phase with gradient elution at a flow rate of 1.0 mL/min and detection wavelength as 264 nm. An Agilent Zorbax SB-C18 (150 mm×3.0 mm,1.8 μm) column was used in the second chromatography dimension with acetonitrile as the mobile phase at a flow rate of 0.42 mL/min, and detection wavelength as 264 nm. In determining vitamin D 3 content, there was a good linear relationship in the concentration range. The system suitability, recovery rate, repeatability and sample stability all met verification requirements., Conclusions: The two-dimensional liquid chromatography method developed in this study is accurate, reproducible and simple, and can simultaneously separate pre-vitamin D 3 , trans-vitamin D 3 , vitamin D 3 , and tachysterol D 3 .