Among the most critical components in neuronal interfaces is the implanted electrode which requires the long-term stability of its electrical performance and biocompatibility of electrode material in contact with live neuronal cells. Reduced graphene oxide (rGO) renowned for its high electrical conductivity and optical transparency has shown great potential for a variety of applications such as transparent conducting electrodes and biosensors, and might be a potential candidate material for the next-generation neuronal interfaces. However, there have been only few systematic studies on graphene-based neuronal interfaces in terms of electrical conductivity and biocompatibility. In this report, we maintained rat hippocampal neurons on top of the rGO multilayers and observed that the viability of neurons is minimally affected and comparable to those grown on a glass substrate up to 30 days in vitro. These results implicate that rGO multilayer can be utilized for excellent neuronal interfaces with its high electrical conductivity and biocompatibility.