A new continuous cell line designated as SGA has been developed from the skin tissue of the freshwater mosquitofish Gambusia affinis. The cell line grew well in Leibovitz's L-15 medium supplemented with 15% fetal bovine serum at 28 °C. Immunophenotyping of the cell line showed the epithelial nature of the cells. Chromosome number analysis showed that SGA cells have a modal diploid chromosome number of 48. Replication of the two different strains of betanodavirus (RGNNV and SJNNV) in SGA cell line showed the maximum virus titre of 108.82 TCID50 mL−1 for RGNNV and 107.2 TCID50 mL−1 for SJNNV. The cytopathic effect in the cell line was observed at 3 days post-infection (dpi) and multiple vacuolations were observed at 7 dpi. Further, DsRed2 plasmid was efficiently delivered into SGA cell line and was found that these cells were transfectable and can be used for assessing gene promoter activity. In vivo challenge experiments using the RGNNV infected cell culture supernatant showed signs of the disease in healthy mosquitofish with mortality commencing from 15 dpi. The above results suggest that the cell line is permissive for propagating betanodavirus and also could be an essential tool for studying the molecular pathogenesis of betanodavirus infection. © 2019 Elsevier B.V.