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Olfactory ensheathing cells (OECs) are one of the most promising candidates for cell-based therapy in repairing spinal cord injury (SCI). However, the conventional method of transplanting OECs, orthotopically or intrathecally, leads to a low rate of cell replacement and limited utilization due to the impediments of chemical and physical barriers after SCI. In this study, we fabricated a three-dimensional (3-D) silk fibroin (SF) scaffold with a novel water-rinsing process. Compared to traditional methanol-treated scaffolds (MTS) and ethanol-treated scaffolds (ETS), the present water-rinsed scaffold (WRS) had a biomimetic nanofibrous structure and softer mechanical properties and was shown to be more biocompatible with OECs. The in vitro study showed that the 3-D SF scaffolds supported OEC adhesion and spreading and that the OECs had a normal cell phenotype. A quantitative study using the CCK8 assay revealed that OEC proliferation on the WRS was higher than that on the MTS and ETS. Moreover, a significantly higher level of GDNF was detected when the OECs were cultured on the WRS. Collectively, these results suggested that the 3-D biomimetic SF porous scaffolds not only provide suitable microenvironments for governing OEC behaviour but also serve as a promising transplantation strategy for OECs to repair SCI.