No.2 of Xiangyun crucian carp possesses the advantages of sterility, rapid growth and improved disease and stress resistance. In order to explore the molecular mechanism of its disease resistance, triploid hybrid interferon a3 (3nIFNa3) has been cloned and characterized. The CDS of 3nIFNa3 comprised 552 nucleotides, encoding 184 amino acids. The first 23 amino acids of N-terminal were signal peptides. There are 2 cysteine residues involved in the formation of disulfide bonds in mature peptides of 3nIFNa3, which indicates that 3nIFNa3 belongs to group I type I interferon. qPCR results showed that the mRNA levels of 3nIFNa3 in the host cells reached the peak at 8 h after poly (I: C) stimulation and reached the highest at 48 h after GCRV or SVCV infection. Both Western blot and immunofluorescence results showed that 3nIFNa3 was a secretory protein and its molecular weight was around 21.8 ku, which was mainly distributed in the cytoplasm before secretion. Further studies showed that the transcription of endogenous ISG genes were significantly up-regulated after the host cells overexpressed 3nIFNa3 or were incubated with the 3nIFNa3-containing conditioned media. The transcription levels of 3nSTAT1, 3nVIPERIN or 3nPKR reached the highest at 2, 2 or 4 h after the host cells were incubated with the 3nIFNa3-containing conditioned media. Besides, the results of the classic plaque assay and the crystal violet staining showed that EPC cells presented significantly enhanced antiviral ability against GCRV and SVCV after incubation with 3nIFNa3 or overexpression of 3nIFNa3. The above results show that 3nIFNa3 is a secretory cytokine and plays an important role in the host antiviral innate immune response.