The invention discloses a preparation method of amphoteric starch size. The method comprises the following steps: preparing an ATS (amylomaltase-treated starch) water solution with mass fraction of 20-50%, regulating the pH value of the ATS water solution to be 3-5, carrying out heating, stirring, nitrogen pumping and other treatment processes, dropwise adding 10-20g of grafted monomer, adding Fe2<+>-H2O2 as an initiator, reacting for 1-3 hours under the protection of nitrogen, adding 1.6-5mL of hydroquinone solution with mass fraction of 2%, regulating the pH value of the reactant to be 4-7 with 1-3mol/L of NaCO3 solution and then carrying out filtering, vacuum drying, grinding, sieving and other treatment processes, thus obtaining the product, wherein the mole ratio of DAC to sodium acrylate in the grafted monomer is (0.5-2) to 1. The amphoteric starch size disclosed by the invention has the advantages of simple process, high sizing viscosity, easiness in desizing, and the like, and is mainly applied to the field of warp sizing.