Interferon, an important cytokine, is an immunomodulator and possesses antiviral and anti-tumour activity. In vitro, it can be administrated in the treatment of diseases alone or with genetically engineered vaccine to enhance the immune effect of the latter. The recombinant transferring vector pSY681–ChIFN-γ was obtained in this study by inserting the chicken type II interferon (ChIFN-γ) gene into the Fowlpox virus (FPV) transferring vector pSY681. The resulting plasmid was then transfected into chicken embryo fibroblast (CEF) cell cultures pre-infected with the parental FPV S-FPV-017. Finally, the recombinant Fowlpox virus (rFPV) expressing ChIFN-γ (rFPV–ChIFN-γ) was produced by homologous recombination with the FPV gene in CEF. rFPV-positive plaques were verified by polymerase chain reaction (PCR), restriction analysis and indirect immunofluorescence assays. The rFPV–ChIFN-γ supernatants, cultured in CEF for 72 h and inoculated into rat fibroblasts (L929), had an inhibitory effect on the replication of Rous sarcoma virus (RSV) with an antiviral titre of 2048 U/ml.