The invention relates to a preparation method of amphoteric ion modified ultra-fine iron oxide particles. The preparation method comprises the following steps: dissolving trivalent ferric salt in solvents, adding sodium citrate, stirring, adding anhydrous sodium acetate, stirring, reacting for 3 to 4 hours at the temperature of 190 to 200 DEG C, cooling, carrying out centrifugation and drying to obtain ultra-fine Fe3O4 nanoparticles, then dispersing the nanoparticles in ultrapure water, performing ultrasound, and EDC and NHS activation, dropwise adding to the ultrapure aqueous solution of Mal-PEG-NH2, reacting for 60 to 72 hours to obtain an aqueous solution of Fe3O4-PEG-Mal, and then dropwise adding the ultrapure aqueous solution of L-cysteine, reacting for 60 to 72 hours, dialyzing and freeze-drying to obtain the amphoteric ion modified ultra-fine iron oxide particles. The method disclosed by the invention is simple, and the prepared nanoparticles are long in half-life periods of blood in mouse bodies, and can realize blood pool angiography in animal levels and enhance imaging for subcutaneous transplanted tumors T1 of HeLa cells, so that the preparation method of the mphoteric ion modified ultra-fine iron oxide particles has potentials of industrialization and commercialization.