Abstract: In the last decades, the marine cage-cultured large yellow croaker (Larimichthys crocea) in Fujian province suffered from Megalocytivirus frequently. The Megalocytivirus isolated from L. crocea have not found any sensitive cell-culture model yet. The missing of culture methods has set back the studies on Megalocytivirus from L. crocea. The Siniperca chuatsi cell line mandarin fish fry cell line-1 (MFF-1) derived from S. chuasti fry, which has been proved to be highly sensitive to multiple ISKNV-like and RSIV-like Megalocytivirus members, may also be a promising culture system for L. crocea Megalocytivirus. To establish methods for cell culture and classification of Megalocytivirus strains from L. crocea, and to make a comparison of their major capsid protein (mcp) genes, which will benefit for the studies of invasion and prevention of these unclassified Megalocytivirus, kidney tissue homogenates of Megalocytivirus-positive L. crocea juveniles (FD201807 and SA201808) were inoculated to the MFF-1 cell line and subcultured continuously. From the tissue homogenates and freeze-thawed infected cells, the virus genome was extracted. The virus mcp was then cloned and sequenced, and compared with the NCBI GenBank records of Megalocytivirus, and a 2018–2020 Fujian collection of 15Megalocytivirus isolates from L. crocea as well. The results showed both two Megalocytivirus isolates caused typical cytopathic effects (CPE) on MFF-1 cells after 3 passages, of which the key features included cell rounding and shrinking, increased cell diopter, continuous cell detachment and particulates secretion with time. Hexagonal viral particles and empty capsids with a size of 130-150 nm were observed in the cytoplasm of infected MFF-1 cells under a transmission electron microscope (TEM). With the processing of virus subculture, the CPE interval of FD201807 shortened from 10 d to 3-5 d, while which of SA201808 remained 7-8 d. mcp gene revealed a 21-bases difference between SA201808 and FD201807. Phylogenetic and clustering analysis indicated that the mcp gene of SA201808 was highly homologous to LYCIV-Zhoushan (GenBank: MW139932. 1), while the homological identity of mcp gene between FD201807 and lateolabrax maculatus iridovirus (LMIV, GenBank: MH577517. 1) was up to 99. 93%. 12 of the total 15 Fujian L. crocea Megalocytivirus isolates were found clustered with SA201808, and the other 3 were clustered with FD201807. In this study, L. crocea Megalocytivirus were isolated through MFF-1 cell culture, indicating the differences among L. crocea Megalocytivirus strains, which could benefit for better understanding of this virus group.