Abstract: The present disclosure provides a recombinant herpesvirus of turkeys (HVT) and a preparation method and use thereof. The present disclosure specifically provides a recombinant HVT, where an exogenous gene is inserted in a spacer region between an HVT005 region and an HVT006 region of an HVT genome; and the exogenous gene is selected from a gene derived from the group consisting of a Newcastle disease virus (NDV), an avian influenza virus (AIV), and an infectious bursal disease virus (IBDV); the spacer region between an HVT005 region and an HVT006 region of an HVT genome is located between 8,867 nt and 9,319 nt of the HVT genome, and has a nucleotide sequence set forth in SEQ ID NO: 1.