The invention discloses a preparation method of standard substances of cytokine ELISA. The preparation method includes the steps of S1, precooling relative equipment for freeze drying until the temperature drops to 4 DEG C; S2, after dissolving standard protein of the cytokine in water, using a freeze-drying solution to dilute the dissolved protein until the concentration of the protein reaches the target concentration, and then obtaining a protein solution with standard concentration; S3, subpackaging the protein solution with the standard concentration into a precooled Sieling bottle, and gently putting on a cover; S4, placing the Sieling bottle containing the protein solution obtained in the step S3, in a vacuum freeze-drying machine until the protein solution is completely dried, and then obtaining the standard substances of the cytokine; the freeze-drying solution in the step S2 is prepared from, by mass, 0.5-5% of sodium caseinate, 1-10% of mannitol, 0.05-0.5% of polypyrrolidone K30 and the balance phosphate buffer with concentration of 0.01 mol / L. By using the preparation method, the standard substances of the cytokine, which have a two-year service life, can be obtained. By adopting the formula of the freeze-drying solution in the method for freeze drying, a great molding effect can be achieved, the appearance of the product is good, and undesirable phenomena such as wall hanging can not occur.