The invention discloses a Muscovy duck parvovirus VP3 genetic recombination fowl pox virus transfer vector and a building method thereof. The method includes: fowl pox virus genome is used as the template to amplify the homologous recombination arms TKL and TKR of TK genes, the TKL and TKR are connected into a TK fragment with multiple cloning sites (MCS), and the TK fragment is connected into a framework plasmid to obtain the plasmid pTK; a reverse serial connection expression box S is synthesized and inserted into the plasmid pTK to obtain the plasmid pTKS; VP3 genes are inserted into an MSC1 position behind an early promotor P7.5, and EGFP BGH pA is inserted into an MCS2 position behind a late promotor P11 to obtain the target transfer vector pTKS-VP3-EGFP. After the pTKS-VP3-EGFP transfects chicken embryonic fibroblast infected by the fowl pox virus, RT-PCR and fluorescent protein detection show that VP3 can be expressed normally. By the Muscovy duck parvovirus VP3 genetic recombination fowl pox virus transfer vector and the building method thereof, a foundation is laid on the further development of safe and efficient MDPV recombinant fowl pox virus vaccines.