Orf is a highly contagious zoonotic disease of small ruminants caused by Parapoxvirus. Kisspeptin, encoded by the KISS1 gene with its cognate receptor GPR-54 is recognized as an upstream orchestrator in the hypothalamic-pituitary-gonadal axis. This study was designed to construct a DNA vaccine that produces a fused peptide composed of a major immunodominant protein of the orf virus (B2L) and kisspeptin-54, a neuropeptide with recognized roles in mammalian reproductive biology. The administration of this recombinant vaccine is shown to produce a significant antibody and cell-mediated immune response directed against B2L compared to the control group (p < 0.05). Furthermore, we found that rats inoculated with PBK-asd vaccine up-regulated antigen-mediated splenocyte proliferation and significantly raised antigen-specific tumor necrosis factor-alpha (TNFα-), interferon-gamma (IFN-ϒ) and interleukin (IL-2) compared to the control group (p < 0.05). This recombinant vaccine also stimulated antibody responses to kisspeptin and decreased serum luteinizing hormone and testosterone levels. Moreover, the current recombinant vaccine caused testicular atrophy and arrested spermatogenesis. It is concluded that this recombinant B2L and Kisspeptin-54 vaccine could be a promising approach for construction of bivalent orf virus and immunocastration vaccine. Furthermore, we concluded that the orf virus envelope protein (B2L) could be used as an immunomodulator for kisspeptin-54 to produce a strong antibody response.