To obtain a continuous monoclonal Oreochromis niloticus macrophage cell line, macrophages were isolated and purified from peritoneal cavity of O. niloticus. The O. niloticus macrophage cell line was established and EBV infection identification, electron microscopy, telomerase activity assay, carcinogenicity evaluation, karyotype analysis, and molecular biology identification were then carried out. The results showed that EBV had been integrated into O. niloticus peritoneal macrophages and stably expressed. After 30 generations of stable passage, the cell line still maintained a good proliferative state. The cell line surface was not smooth and had obvious blunt round protrusion and slender pseudopodia, which was the typical morphology performance of the macrophages. Telomerase activity of this cell line was significantly higher than that of untreated macrophages (P<0.05), but the difference was not significant with HeLa cells, and the cell line was not carcinogenic, which indicated that immortalized cell lines were successfully constructed. Karyotype analysis showed that the cell line had 44 chromosomes, and the karyotype formula was 2 n=2 x=44=4 sm+17 st+1 t. PCR detection revealed that CD33, and CD205 transcripts were found in this cell line, all of which were markers of monocytes/macrophages. 18S rRNA detection showed that the cell line was from O. niloticus macrophages. Immortalized tilapia macrophage cell lines were successfully established. The establishment of this cell line laid the foundation for the further study of the immunological function of peritoneal macrophages in O. niloticus and also provided a tool for studying the mechanism of high protection rate of O. niloticus Streptococcus HSP70-peptide vaccine.