Abstract: OBJECTIVE To construct the transgenic mice model expressing tick-borne encephalitis virus (TBEV) NS5 protein, so as to provide a platform for the evaluation of RdRp enzyme activity during TBEV infection. METHODS TBEV NS5 transgenic mice were generated by microinjection of the linearization vector of pRP.ExBi-EF1α-NS5-IRES-eGFP into FVB/N mouse fertilized eggs, which were then transferred into pseudopregnancy mice to give birth to transgenic founder mice. Subsequently, homozygous transgenic mice with genetic stability were selected by breeding. The expression of TBEV NS5 gene was examined. RESULTS Five TBEV NS5 transgenic founder mice were screened and bred to the fifth filial generation(F5). TBEV NS5 gene was identified by PCR, RT-PCR and Western blot. CONCLUSION The transgenic mouse model expressing TBEV NS5 was generated successfully.