The invention discloses a preparation method and the production process of a complex injection of 13 vitamins. The injection adopts dual-injection type, the invention overcomes the problem of solubility of the fat-soluble vitamins in water, so the invention can be prepared into the water injection; the stability of the product is good, and various vitamins can not be affected mutually. The injection includes water-soluble vitamins, fat-soluble vitamins, surfactant, antioxidant, complexing agent and solvent, the injection water is added till the dissolution, the activated carbon is added for absorption, the pH value of the solution is regulated, and then the nitrogen filling and sterilization are carried out. The production process of the invention prepares the injection into dual-injection type, the vitamin B12, folic acid and biotin have an independent package, the process overcomes the problem of solubility of fat-soluble vitamins in water, the invention can be prepared into water injection, the stability of the product is good, and various vitamins can not be affected mutually, the vitamins can be added in a large infusion for usage, so as to avoid the trouble of redissolution and reuse of the freeze-dried powder injection.