The invention discloses a high performance liquid chromatographic analysis method for simultaneous determination of the content of 14 kinds of vitamins; octadecylsilane bonded silica gel is used as a filling agent, a 0.1 M sodium acetate solution-methanol is used as a mobile phase for gradient elution, gradient flow velocity of the mobile phase and a program change wavelength of an ultraviolet detector are combined, and intact separation and quantitative analysis of the 14 kinds of vitamins (including 4 kinds of fat soluble vitamins and 10 kinds of water soluble vitamins) can be achieved in 60 minutes. The method has the advantages of strong specificity, high precision, good stability, high accuracy, simple operation, and cheap and easily available reagents and consumable materials, is suitable for quality control of compound vitamin products in medicine and food industries, and can effectively save the cost and time of detection.