In this study, we provide the first comprehensive annotation of the entire family of canine interferons (IFNs). Canine IFN-ɛ (IFNE), IFN-κ (IFNK), and IFN-λ (IFNL) were discovered for the first time. Ten functional and 2 truncated IFN-α (IFNA) pseudogenes were found in the genome, which also enriched the existing knowledge about canine IFNA. The canine type I IFN genes are clustered on chromosome 11, and their relative arrangements are illustrated. To further investigate the biological activity of canine IFNE, it was expressed and purified in Escherichia coli. Recombinant canine IFNE (rCaIFN-ɛ) displayed potent antiviral activity on both homologous and heterologous animal cells in vitro, indicating that rCaIFN-ɛ has more broad cross-species activity than recombinant canine IFNA (rCaIFN-α). The antiviral activities of rCaIFN-ɛ and rCaIFN-α7 against different viruses on MDCK cells were also evaluated. The antiviral activities of recombinant canine IFNK and IFNL were demonstrated using a VSV-MDCK virus-target cell system. rCaIFN-ɛ exhibited a significant anti-proliferative response against A72 canine tumor cells and MDCK canine epithelial cells in a dose-dependent manner. rCaIFN-α7 was approximately 16-fold more potent than rCaIFN-ɛ in promoting natural killer cell cytotoxicity activity. Further, rCaIFN-ɛ can activate the JAK-STAT signaling pathway.