A method for simultaneously determining fat-soluble vitamins and carotenoids in serum, and belonging to the technical field of analytical chemistry, includes an ionic liquid (IL) or a binary mixed solvent composed of an IL and another solvent is adopted as an extractant; the biological samples are pre-treated by liquid-liquid extraction (LLE) and then detected by high-performance liquid chromatography (HPLC); retinyl acetate and trans-β-apo-8′-carotenal are adopted as the internal standards for fat-soluble vitamins and carotenoids, respectively; and the internal standard method is adopted to establish standard curves for quantification based on the retention time and the UV-Vis absorption spectrum. Compared with the existing methods, in the disclosure, the pretreatment process is simple and easy to be conducted, the sample can be prepared in a short time, and the toxic and harmful organic solvent is used at a reduced amount.