Objective To investigate the possibility of culturing human oral keratinocyte using autologous serum in order to provide theoretical and technical foundation for clinical application of tissue engineering oral mucosa epithelium.Methods The human oral keratinocytes were cultured by the medium containing different concentrations of autologous serum(10%,20%,30%)and fetalbovine serum (10%), respectively. The growth conditions for the cell and the mucosa epithelium in the groups were observed, the cell growth curves were drawn, and the population doubling time (PDT) was counted. Results The results showed that the human oral keratinocyte could proliferate well in the medium containing autologous serum or fetal bovine serum. The differences in the 24hour clone rate and PDT were not significant. Both the area and the thickness of the cultured oral epithelium increased with the increase of the autologous serum concentration, and the difference between autologous serum and fetal bovine serum was significant, especially with the medium containing 20% autologous serum( P＜0.05) . The human nature of the cultured epithelium was demonstrated by the immunofluorescent mouse anti-HLA antigen. Conclusion The autologous serum can replace the fetal bovine serum to culture the oral keratinocyte well, and the cultured oral mucosa epithelium can be better differentiated in the autologous serum than in the fetal bovine serum.