Abstract: Various buffers and culture mediums designed originally for mammalian cells or insect cells are widely used for the experiments of crustacean cells. However, the osmolality of crustacean humoral fluids is very different from that of mammals, it is not appropriate to directly use the buffer system of mammalian cells for crustacean cells. In this study, the osmolality of the serums of Cherax quadricarinatus, Procambarus clarkia and Litopenaeus vannamei (maintained in salinity 30), was measured as (409±12) mOsmol/kg, (465±8) mOsmol/kg and (704±15) mOsmol/kg, respectively. Subsequently, the osmolalities of anticoagulant and PBS buffer were changed by adjusting the concentration of NaCl according to the serum osmolality of the corresponding animal. After optimization, the concentrations of NaCl in anticoagulant and PBS for C. quadricarinatus were 70 mmol/L and 200 mmol/L, respectively, and for P. clarkii were 100 mmol/L and 230 mmol/L, separately. Meanwhile the concentrations of NaCl in anticoagulant and PBS for L. vannamei were 220 mmol/L and 360 mmol/L, respectively. We further compared the haemocyte survivals of these crustaceans in different osmolality buffers. It showed that the mortality rates of hemocytes of C. quadricarinatus, P. clarkii, and L. vannamei reached 4.5%, 2.0% and 10.0%, respectively, when treated in PBS for 15 mins. While in optimized PBS, the rates were reduced to about 1.0%, 1.0% and 2.0%, respectively. However, the mortality rate of haemocyte in anticoagulants was lower (< 2.0%) and there was no significant difference before and after optimization. In conclusion, the optimized buffer solution can improve the state of crustacean cells and the exactness of experiments.