Abstract: In the present study, the optimal conditions for isolation and culture of the primary hepatocytes from liver of Cyprinus carpio were explored.The condition for digestion reaction with trypsin was optimized first and the results from trypan blue staining indicated that the mortality of primary hepatocytes was the highest after treatment of fish liver tissue with trypsin at 25℃ for 40 min.Four different purification and culture conditions were then set up with crude liver cell suspension after trypsin digestion:1.Cells were directly incubatedin L-15/DMEM medium containing 10% fetal bovine serum (FBS);2.Cells were incubated with L-15/DMEM medium containing 10% carp serum;3.Cells were purified using Percoll density gradient centrifugation and then cultured in L-15/DMEM medium containing 10% fetal bovine serum;4.Cells were purified with Percoll and then cultured with L-15/DMEM medium containing 10% carp serum.The results from the morphology by HE staining revealed that the purification by Percoll significantly improved the purity of hepatocytes.In addition, the replacement of bovine serum by carp serum significantly increased the cell proliferation based on the MTS/PMS results.This study will lay the foundation for the establishment of a stable primary hepatocyte model for further toxicological research.