In this paper, the hydro-thermal synthesis method has been used to prepare the three-dimensional self-assembly graphene (3D-G). Different shapes of 3D-G have been obtained by adjusting the reaction-time (6h, 12h, 18h and 24h). And the cytocompatibility of the 3D-G was also evaluated in vitro. And then, SEM, FTIR, Raman and XPS have been used to characterize the microstructure and component of 3D-G. For comparison, the in vitro cytocompatibility of the 3D-G was assessed using mouse fibroblast cells (L929) by MTT. The results showed that the length and diameter of macroscopic 3D-G were decreased, and the mechanical property was increased as the reaction-time increase. In addition, the SEM images showed that the diameters of pores in 3D-G declined and the structure become dense with the increasing of reaction-time, which means the defects of 3D-G decreased and the structure orderly. Meanwhile, the state of cell adhesion, growth and proliferation observed on 3D-G with the reaction time of 24 h was the best during the culture, which means it was available for biological application.