Abstract: A stable cell culture system in vitro can provide a platform for the study of gene function and host-pathogen interaction. In this study, the culture conditions of hematopoietic tissue (HPT) cells of crayfish (Cherax quadricarinatus) were optimized to generate stable HPT culture with high viability. After isolating and extracting HPT cells from crayfish, we compared the culture conditions, such as culture medium (L-15, Express Five and DMEM), glutamine concentration (0, 1%, 5% and 10%), fetal bovine serum concentration (0, 1%, 5% and 10%), and culture temperature (20 ℃ and 27 ℃). Results of cell morphology observation and cell viability analysis showed that the cell culture of L-15 medium was optimized by supplemented with 1% penicillin-streptomycin, 10% L-glutamine and 1% fetal bovine serum, in which the cells could survive for about 30 days at 20 ℃. The culture conditions are helpful for in vitro culture of hematopoietic tissue cells of crayfish.