Synthesis and three-dimensional (3D) assembly of magnetite nanocrystals were realized by a one-pot procedure, in which Fe(acac)(3) (acac = acetylacetonate) was partly reduced by hydrazine accompanied with ethylene glycol and spontaneously assembled into spherical nanostructures in the presence of surfactants including beta-cyclodextrin, oleic acid, and oleylamine. The size of the assembled spheres can coarsely be controlled in a limited range (100 nm to 2 mu m) by changing the reaction temperature and the concentration of beta-cyclodextrin. X-ray diffraction and far Fourier transform infrared spectroscopy were employed to clarify the structures of magnetite in the assembled spheres. Electron diffraction pattern in a selected-area exhibits a high-crystallinity characteristic of cubic structure magnetite. We found that the formation of spherical magnetite aggregates highly depends on the presence of beta-cyclodextrin, while oleic acid and oleylamine improve the morphology of individual magnetite nanoparticles in the assembled spheres. In addition, the thermal gravimetric analysis and differential thermal analysis were applied to determine the content of magnetite in the products. Magnetic properties were also studied by using a superconducting quantum interference device magnetometer.