The invention provides a composite vitamin injection pharmaceutical composition containing 13 vitamins and a preparation method thereof. The composition comprises 4 fat-soluble vitamins and 9 water-soluble vitamins, which are respectively placed in two ampoules, wherein the first ampoule contains vitamin A palmitate, vitamin K1, vitamin D3, vitamin E, vitamin C, vitamin B1, riboflavin sodium phosphate, dexpanthenol, vitamin B6, nicotinamide and a cosolvent, an antioxidant, and a pH adjusting agent; the second ampoule contains vitamin B12, folic acid, biotin, a stabilizing agent and a pH adjusting agent. The preparation method comprises that the preparation of the first ampoule comprises steps of oil-phase medicinal liquid preparation, water-phase medicinal liquid preparation, mixing, pH adjustment, filtration, filling, sterilization, and the like, and the preparation of the second ampoule comprises steps of medicinal liquid preparation, pH adjustment, active carbon adsorption, decarburization, filtration, filling, fusing sealing, sterilization and the like. According to the invention, the solubilizer, the antioxidant, and the preparation process of the composite vitamin injection pharmaceutical composition containing 13 vitamins are improved, and the composite vitamin injection pharmaceutical composition has the advantages of few using kinds of auxiliary materials, good stability, high clarity, high security, simple preparation method, and easy realization of industrialization.