The invention belongs to the field of analytical chemistry, and particularly relates to a method for separating and determining a calcipotriol intermediate F and impurities thereof. According to the method, solid-liquid separation is performed by adopting octadecylsilane chemically bonded silica as a chromatographic column of a filling agent and taking a mixed solution of methanol and ethyl acetate as a mobile phase, and the impurities comprise one or more of VD2, E, D and an isomer F' of F; effective separation of the intermediate F and the enantiomer impurity thereof can be achieved, effective separation of the intermediate F and the other impurities also can be achieved, the content of the intermediate F and the impurities thereof can be further accurately determined, and the specificity and the sensitivity are high. The operation method is simple, has the advantages of being simple, convenient and rapid and has the extremely important significance on quality control and safety guarantee of the calcipotriol intermediate F.