Objective To explore how to fabricate a innovative silk fibroin nerve conduits (SF-NCs) with linearly oriented multiple-channel structure. Methods The innovative SF-NCs was fabricated by a vertical sequential cooling-thermal induced phase separation (TIPS) processing, its morphology was observed by Scanning electron microscopy (SEM), MTT assay was used to quantitatively analyzed the PC12 cells viability co-cultured with the innovative SF-NCs, SEM was used to observe the adhesion and morphology of PC12 cells seeded into the innovative SF-NCs, PC12 cells were used to assess the NGF bioactivity released from the SF-NCs. Results The SEM results showed that the new fabricated SF-NCs had linearly oriented lamellar-like multiple-channel, which distributed evenly. The spaces between parallel lamellar-like channels (ds), porosities and compressive strengths (σc, Ec) of the SF-NCs decreased with decreasing T ft . MTT assay results showed that the viability of PC12 cell was better than the control group (P < 0.05). The SEM observation indicated that PC12 cells showed good adhesion and differentiation with neuritis outgrowth during the period of co-culture with the SF-NCs. NGF release from the innovative SF-NCs was prolonged over 4 weeks, and remained bioactive. Conclusion The new fabricated SF-NCs with special topography structure, excellent mechanical properties and good biocompatibility. Importantly, the SF-NCs are also biomimicking the anatomy structure of peripheral nerve fasciculus and could be used as another alternative of artificial nerve conduits.