The invention relates to a mobile phase for separating phosphatide by a highly effective liquid phase chromatography. The components of the mobile phase comprise 10 percent to 50 percent of methyl alcohol, 50 percent to 85 percent of acetonitrile, 0.5 percent to 3 percent of fluorinated organic acid, and 0.5 percent to 3 percent of water. Through the long-term operation, the mobile phase of the invention cannot result in any deposit, cannot damage a pump system, and has no damage to the pump system of a liquid phase chromatographic instrument; the mobile phase can be used to detect the separated phosphatide in an evaporative light-scattering detector, compared with an ultraviolet detector, the base line detected by the evaporative light-scattering detector is stable, the quantitative analysis is easy, the sensitivity is high, and the application of the mobile phase has no limit; compared with the gradient method, the method of the invention uses a single pump to realize the separating detection; when the mobile phase adopts the appropriate compounding ratio, besides the phosphatide can be separated, the unknown materials that cannot be separated by the other mobile phases can also be separated. When the mobile phase of the invention applies to the isocratic eluting, the operation is simple and time-saving, the reproducibility is good, and the duplicity is easy.