Ginkgo biloba extract (GBE) has been widely used for treatment of neural damage and disorders. Neural stem cells (NSCs) hold promise as a treatment of hearing loss caused by neural damage. However, the biological functions of GBE in modulating NSC behaviors in the cochlea are still largely elusive. In this study, we sought to explore the effects of GBE on the differentiation and performance of NSCs from mouse cochlea. Our data showed that GBE treatment promotes cell survival and NSC proliferation. In addition, GBE treatment also increases NSC differentiation to neurons and enhances the performance of mature neural networks evident by the increased frequency of calcium oscillation. Moreover, neurite outgrowth is also dramatically increased upon GBE treatment. Overall, our study demonstrates the positive regulatory role of GBE in NSC proliferation and differentiation into functional neurons in vitro, supporting the potential therapeutic use of GBE in hearing loss recovery.