Gene therapy and nerve stem cells isolated from the developing human enteric nervous system (ENS) are significant. They may open the route for the cell therapy of Hirschsprung's disease (HD). We have constructed the recombinant adenovirus-carrying glial cell line-derived neurotrophic factor (GDNF) and endothelin receptor B (EDNRB) gene, and investigated the exosomatic coexpression in neural stem cells. The recombinant adenovirus Ad-GE coexpressing GDNF and EDNRB gene was constructed by the AdEasy system and confirmed by the reverse transcription polymerase chain reaction (RT-PCR) method. Expression of exogenous genes in neural stem cells after transfection was confirmed by the flow cytometry and real-time fluorescence quantitative PCR. Fragments of pAd Track-CMV-GE were consistent with GDNF and EDNRB. The green fluorescence of the positive cells was followed by fluorescence microscopy at 24 h after NSCs had been transfected, reaching a peak at 72 h after transfection. Flow cytometry showed that the efficiency of transfection was 15.0, 23.6, and 25.4% at 24, 48 and 72 h respectively. Real-time fluorescence quantitative PCR showed the expression levels of mRNA of GDNF and EDNRB in 48 and 72 h groups were obviously higher than that in 24 and 96 h groups. Recombinant adenovirus carrying GDNF and EDNRB genes are coexpressed in neural stem cells, which may offer the possibility of a novel approach to local combination gene therapy for Hirschsprung's disease.