The invention discloses a mixed fatty acid high performance liquid phase chromatography method and application thereof. In order to solve the problems that the prior art has single method in separating linolenic acid, palmitic acid, linoleic acid and other three fatty acid mixtures with very similar chemical and physical properties with HPLC, and defects in elution method, mobile phase composition selection and the like, the invention provides a method for simultaneously separating six fatty acid mixtures, i.e. linolenic acid, palmitic acid, linoleic acid, oleic acid, palmitic acid and stearic acid with HPLC. The method esterifies fatty acid solution to be tested with the acetone solution of omega-bromoacetophenone and the acetone solution of triethanolamine, and separates the solution with HPLC. The HPLC adopts an isocratic elution method, and the mixture of acetonitrile and CTAB solution or acetonitrile-water solution is selected to serve as the mobile phase. Compared with the priorart, the invention has the advantages of simple and feasible operation, good separation effect, high detection sensitivity, accurate and reliable results, and simultaneous separation of 6 fatty acids.