The invention discloses a method for simultaneous determination of various fat-soluble vitamins in serum by using a semi-automatic sample treatment liquid chromatographic technology; the HPLC method is adopted, and the chromatographic conditions comprise that a mobile phase A is a 0.05% trifluoroacetic acid aqueous solution, a mobile phase B is a 0.02% trifluoroacetic acid methanol-ethanol mixed solution, and gradient elution is adopted; the various fat-soluble vitamins are selected from vitamin A, vitamin D2, vitamin D3, alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, K1, K2 or Q10. Through optimization of a sample pretreatment method and high performance liquid chromatographic conditions, the method for simultaneous determination of the various fat-soluble vitamins in the serum is established, can carry out precise qualitative and quantitative analysis, is a detection method having the advantages of simple and fast sample processing, high flux, reliable result and low cost, has the advantages of strong specificity, high sensitivity, simple operation, low cost and the like, has easily analyzed and objective results, and is especially suitable for clinical popularization and application.