Interferons (IFNs) were first discovered in 1957 in a nutrient fluid from chick chorioallantois membranes, where it was observed that administration of virus stimulated interferon production in many animals, tissues, and cells, within a short time. They are classified into type 1, type 2, and IFN-like cytokines, with type 1 IFN classified into IFNα, IFNβ, IFNε, IFNκ, IFNω, IFNδ, and IFNτ. Clinical tests for recombinant human IFNs and bovine IFNτ have been conducted since 1981. Although infections of Highly Pathogenic Avian Influenza Virus (HPAI) have continued to cause high economic losses in poultry industry causing many deaths of poultry, few molecular experiments based on gallus (ga) IFNs have been reported since 1994 and clinical trials to test their use are limited. Here, we examined the activities of newly produced three recombinant gaIFNα3s on different species of cells. The recombinant gaIFNα3s showed significant antiviral activity in Gallus embryo fibroblast (GEF) cells, showing good potential to prevent the cytopathic effect of vesicular stomatitis virus (VSV). However, they failed to protect Wistar Institute Susan Hayflick (WISH) cells, Madin-Darby bovine kidney epithelial (MDBK) cells, and Madin-Darby canine epithelial-like (MDCK) cells. This study demonstrated the impact of species specificity on the antiviral activity of gaIFNα3 and the effect of location of fusion protein.