The invention relates to a preparation method Fe3O4 nanoparticles with a cluster structure. The preparation method comprises steps as follows: preparation of extra small Fe3O4 nanoparticles, preparation of an activated extra small Fe3O4 nanoparticle solution, and preparation of the Fe3O4 nanoparticles with the cluster structure. The preparation method is simple, the prepared Fe3O4/Cystamine nanoparticles have good stability and biocompatibility, are sensitive to reducing conditions, can realize T1-T2 dual-mode MR imaging effect of a tumor location in an animal body, can be effectively taken asan MR imaging contrast medium and have industrialized and commercialized application prospect.