Abstract: Interferon-γ is a cytokine crucial for the regulation of both natural and cellular immunity, playing an important role in defending against virus invasion and bacterial infection. In this study, the cDNA of interferon-γ genes was amplified from the spleen tissues of Nile tilapia; this cDNA had an open reading frame of 621 bp, encodeing 206 amino acids. Bioinformatics analysis shows that the interferon-γ molecule of Nile tilapia contains one signal peptide and one nuclear localization signal. Its tertiary structure was similar with the crystal structure of human interferon-γ. Real-time fluorescent quantitative polymerase chain reaction (PCR) shows that the expression level of interferon-γ gene was the highest in the spleen of normal Nile tilapia, followed by the intestines, gills and kidneys (P < 0.05). Both the high expression of interferon-γ in immune-associated tissues and its upregulated expression after GBS infection reveal the significant role of interferon-γ in the immune defense of Nile tilapia. This study lays foundation for further research on the function of interferon-γ gene in Nile tilapia as well as its development and application in transgenesis and selective breeding for disease resistance.