In order to explore the stable and reliable methods for isolation and primary culture of hepatocytes from Epinephelus coioides, we selected the hepatocytes of E. coioides and then cultivated them under different conditions. E. coioides hepatocytes were isolated by tissue separation and trypsin (0.25% with EDTA) digestion. The hepatocytes could be separated and purified by density gradient centrifugation. The harvested hepatocytes were then suspended in DMEM/F-12, M199, or L-15 (cultured with 5% CO₂). The yield was determined by a hemocytometer. The viability was assessed by Trypan blue exclusion test. Proliferation of hepatocytes was tested by MTT assay. Function of hepatocytes was examined according to the levels of lactic acid dehydrogenase (LDH), albumin (ALB), and urea nitrogen (BUN)of supernatant at different time, respectively. Trypsin digestion method was better than tissue culture. Using 0.25% warm trypsin digestion, the cell yield was 1.6×10⁸ cells per g(liver weight) and the viability was more than 95%. The cells growth were better cultured in L-15 medium than in DMEM/F-12 and M199 media. The liver function index showed that lactic acid dehydrogenase (LDH) significantly decreased, urea nitrogen (BUN) and albumin (ALB) significantly increased during 48-72 h with a strong proliferation. This study indicates that the best method of isolation was pancreatin digestion and the best medium was L-15. During 48 to 72 h in culture, the growth and metabolism of hepatocytes were thriving.