Abstract: Objective Rat glial cell derived neurotrophic factor(GDNF) gene and tyrosine hydroxylase(TH) gene are widely used in gene therapy of Parkinson's Disease(PD).The present study is designed to investigate whether the two target genes GDNF and TH could be expressed simultaneously in one adeno-associated virus(AAV)vector and to discuss the probability of using it in gene therapy of PD. Methods RNA of GDNF and TH in HEK293 packaging cells were examined by reverse transcription-polymerase chain reaction (RT-PCR). Expressions of GDNF and TH in infected bone marrow stromal cells (BMSCs) and rat brain coronal sections were examined with immunocytochemistry and immunohistochemistry assay individually. Results RNA of GDNF and TH were detected in HEK293 packaging cells. Expressions of this two genes were observed in both bone marrow stromal cells and brain sections of rats. The expression efficiency of AAV-LacZ was about 50% in HEK293 packaging cells and approximately 15% in infected BMSCs and the expressions of TH and GDNF on the AAV-GDNF/TH virus injected points both had significant differences (p<0.01) compared with opposite injected with PBS. Conclusion Results of the present study suggested that GDNF and TH genes contained in one recombinant AAV vector could be expressed simultaneously both in vitro and in vivo and could provide references for further application in gene therapy of PD.