Fe3O4 nanomaterials have received great attention due to their many applications in tumor diagnosis and tumor heat therapy based on their good biocompatibility, magnetic targeting ability and superparamagnetic properties to avoid magnetic reunion in the process of magnetic targeting. Most of superparamagnetic nanoparticles obtained by traditional methods exhibit lower saturation magnetization (MS), because of their small particle sizes. Enlarging the particle size is favorable to increase the MS of magnetic particles. However, the superparamagnetism of the particle could be lost with the increase of particle size. This is not favorable to the targeting delivery of magnetic particles. For this purpose, in this paper, novel Fe3O4 nano-microspheres with mesoporous hollow structure are successfully synthesized by a facile hydrothermal method from the FeCl36 H2O, sodium citrate, urea, and polyacrylamide as additive, the reaction temperature is 200℃ and reaction time is 12 h. The crystal structure and purity of the resulting products are examined by powder X-ray diffraction (XRD). The morphologies of the products are studied by using scanning electron microscopy (SEM) and transmission electron microscopic (TEM). The magnetic properties of Fe3O4 nano-microspheres are evaluated with a vibrating sample magnetometer. The morphology evolution process and possible formation mechanism of Fe3O4 nano-microspheres are investigated. The findings are as follows:all XRD peaks of the hollow Fe3O4 nano-microspheres could be assigned to the spinel-type Fe3O4. The SEM and TEM images reveal that the products are mesoporous hollow Fe3O4 nano-microspheres and possess hierarchical structure, in which large microspheres (160 nm) are self-assembled by smaller Fe3O4 initial crystals (18 nm). It is found that the synthetic time of Fe3O4 nano-microspheres is considerable for the formation of the Fe3O4 hierarchical structure, and that the dispersion and sphericity of Fe3O4 nano-microspheres are the best when reaction time is 12 h. The formation of hierarchical hollow structure is believed to be due to the Ostwald ripening process, in which the initial crystals redissolve and regrow. Furthermore, the magnetic measurement results show that as-prepared hollow Fe3O4 nano-microspheres exhibit typical superparamagnetic properties whose initial crystal size is in the range of superparamagnetic region. Meanwhile, MS is about 73.3 emu/g at room temperature, which is significantly greater than that of traditional small superparamagnetic nanoparticles and compact solid nano-microspheres. The high saturation magnetization of hollow Fe3O4 nano-microspheres originates from a high crystallinity with primary grain, lager size and hierarchical structure. The results indicate that the as-prepared Fe3O4 hollow nano-microspheres are dispersed, water-soluble, homogeneous in particle diameter, and superparamagnetic, and can be used in targeted anticancer drug delivery and tumor heat therapy.