Abstract: we developed a method to separate the GC and SGC of Cherax quadricarinatus by using discontinuous Percoll density gradient centrifugation. The dilution buffer and the combination of density gradient were optimized. We found that the density gradient should be diluted in anti-clotting buffer to prevent hemocytes aggregation, and the discontinuous Percoll density gradient that consisted of 20%, 65% and 100% of Percoll provided the best result compared with others. After centrifugation at 1 810 r/min for 20 min, the hemocytes were separated into two layers, corresponding to SGC and GC, respectively. Flow cytometry analysis showed that the purities of both fractions were >95% and the cell mortalities were <1.5%. The isolated cells were in good status and could be readily used for further analysis. This simple and effective method will facilitate the study on the function of different hemocytes and thus provide information for disease control.