Spinal cord injury (SCI) is an incurable disease causing an ischemic environment and functional defect, thus a new therapeutic approach is needed for SCI treatment. Vascular endothelial growth factor (VEGF) is a potent therapeutic gene to treat SCI via angiogenesis and neuroprotection, and both tissue-specific gene expression and high gene delivery efficiency are important for successful gene therapy. Here we design the hypoxia/neuron dual-specific gene expression system (pEpo-NSE) and efficient gene delivery platform can be achieved by the combination ex vivo gene therapy with erythropoietin (Epo) enhancer, neuron-specific enolase (NSE) promoter and neural stem cells (NSCs). An in vitro model, NSCs transfected with pEpo-NSE were consistently and selectively overexpressing therapeutic genes in response to neural differentiation and hypoxic conditions. Also, in SCI model, ex vivo gene therapy using pEpo-NSE system with NSCs significantly enhanced gene delivery efficiency compared with pEpo-NSE system gene therapy alone. However, microarray analysis reveals that introducing exogenous pEpo-NSE and VEGF triggers biological pathways in NSCs such as glycolysis and signaling pathways such as Ras and mitogen-activated protein kinase, leading to cell proliferation, differentiation and apoptosis. Collectively, it indicates that the pEpo-NSE gene expression system works stably in NSCs and ex vivo gene therapy using pEpo-NSE system with NSCs improves gene expression efficiency. However, exogenously introduced pEpo-NSE system has an influence on gene expression profiles in NSCs. Therefore, when we consider ex vivo gene therapy for SCI, the effects of changes in gene expression profiles in NSCs on safety should be investigated.