Tilapia is an important aquaculture species world-wide, and China is the largest producer of farmed tilapia. In recent years, alongside the rapid development of tilapia aquaculture in the world, diseases in this fish have become a great concern, including viral diseases caused by beta Nodavirus, herpes simplex virus, and tilapia lake virus, which lead to huge economic losses. So far, research studies on tilapia viral pathogens are dependent on the E-11 cell line derived from grouper and tilapia primary brain cells; thus, a cell line derived from the host of tilapia needs to be urgently established for applications. Therefore, in the present study, by using tissue explant techniques, a novel cell line (designated TiK) derived from the kidney of tilapia (), was established and fully characterized. The TiK cell line are fibroblast-like cells, which have been stably subcultured for more than 50 passages. The optimal growth conditions for the TiK cell line are DMEM medium, 28℃, and 15% FBS. Under the optimal conditions, the doubling time of TiK cells is about 45.8 h. After 6 months of cryopreservation in liquid nitrogen, TiK cells exhibit a viability of about (89.84±3.48)%, as measured by trypan blue staining. Chromosome analysis of TiK cells showed that the number of chromosomes ranged from 20 to 66, and the modal number is 48 at the 32^th passage. The TiK cell line was infected with tilapia virus isolated by our laboratory, and showed typical cytopathic effects, demonstrating that the TiK cell line was susceptible to infection by tilapia viruses. The establishment of the TiK cell line will contribute to research on the control of tilapia viral diseases.