To establish a barfin flounder fin cell line and lay a solid foundation for viral and cytotechnological studies, the fin tissues, digested with hyaluronidase and collagenase II, were cultured in 20% fetal bovine serum(FBS)containing DMEM/F12 (1∶1), M199 and Leibovitz L-15 medium (pH7.2), supplemented with 20% fetal bovine serum, carboxymethyl-chitosan, basic fibroblast growth factor (bFGF) and insulinlike growth factor-I (IGF-I), at 18~26℃, respectively. The results of in vitro culture showed that the optimum medium of the fin cells was DMEM/F12 medium (pH7.2) with the above supplements, and the optimum temperature of them was about 22℃. By utilizing 20% FBS-DMEM/F12 medium with the above supplements, the primary culture of the fin cells was initiated at 22℃. And the fin cells, in fibroblastic morphology, grew rapidly, and formed a confluent monolayer 20 d after culture initiation. By successive subculture, a barfin flounder fin cell line was sucessfully established, and sub-cultured to passage 135 till now. Growth property analysis showed that the fin cells of the cell line at passage 60 had a population doubling time of 56.9h, which indicated that the cells had active proliferating abilities. Chromosome analysis showed that the cells exhibited chromosomal aneuploidy but still had a modal chromosome number of 46 which had a normal diploid karyotype of 1 pair of submetacentric and 22 pairs of telocentric chromosomes. All these indicate that a novel continuous barfin flounder fin cell line was sucessfully established in this study, which as an ideal in vitro researching system, will be of great theoretical and practical significance for cytotechnological breeding and cellvirus interaction studies of barfin flounder.