Abstract: 【Objective】 The expression characteristics among different tissues and the expression pattern of interferon induced protein 44 gene(ifi44) after Streptococcus agalactiae infection and polyinosinic-polycytidylic acidPoly(I:C) challenge were explored in present study to lay a theoretical basis for further researching the function and mechanism by ifi44 in the immune response of fish.【Method】 The open reading frame(ORF) of On-ifi44 gene was cloned and its bioinformatics was analyzed by ExPASy, TMHMM-2.0, Euk-mPLoc 2.0, SOPMA and SWISS-MODEL online softwares. The single-cell RNA-Seq(scRNA-seq) data were employed to analyze the distribution of On-ifi44 gene at the cellular level. The expression distribution of On-ifi44 gene in various tissues of healthy Nile tilapia and the sequential expression after S. agalactiae infection and Poly(I:C) stimulation were detected by real-time fluorescent quantitative PCR.【Result】 The open reading frame of the On-ifi44 gene was 1437 bp, encoding 478 amino acids residues with a protein molecular weight of 52.7 kD and theoretical isoelectric point(pI) of 4.97. On-ifi44 contained a TLDc_dom domain(1-157 aa) and a GTP-bd domain(197-322 aa), but no transmembrane domain. Amino acid sequence similarity between On-ifi44 and Oreochromis aureus ifi44 was 97.94%, phylogenetic tree based on amino acid sequence similarity of ifi44 amino acid sequence similarity showed that On-ifi44 amino acid sequence and O. aureus ifi44 amino acid sequence clustered into a single lineage, which meaned that they had a close genetic relationship. On-ifi44 gene was expressed in 11 tissues of Nile tilapia, including intestine, liver, gills, skin, spleen, kidney, thymus, brain, head-kidney, muscle and heart, and mainly expressed in the T cell subgroup. After S. agalactiae infection and Poly(I:C) stimulation, the expression of On-ifi44 gene was changed significantly in the spleen, kidney, head-kidney, and intestinal, and with obvious time-dependence.【Conclusion】 On-ifi44 contains one TLDc_dom domain and one GTP-bd domain, which is evolutionarily conserved and has similar biological functions among different species. The expression of On-ifi44 gene is up-regulated in a time-dependent manner after S. agalactiae infection and Poly(I:C) stimulation, indicating that ifi44 gene, as an important regulatory factor, is involved in the immune response of Nile tilapia against bacterial infection and viral proliferation.