Serum plays a role as biomarker in the assessment of health condition or severity of diseases. The separation method, storage temperature and duration affect the levels of cytokines and its biochemical constituents. In this study, blood sampling was performed from Hanwoo cattle through jugular venipuncture. All blood samples were kept at 26±3℃ for 1~2 h and then separated into four conditions (groups a-d). For group (a), the serum was immediately separated (control group) and stored at -70℃; for group (b) the separated serum was kept in ice box for 2 h and then stored at -70℃; for group (c) the collected blood samples were stored in ice box for 2hrs before serum separation; and for group (d); the whole blood was kept at 4℃ for 24hrs before serum separation and then stored at -70℃. All serum samples were stored at -70℃ until analysis. Depending on serum separation time, storage period and temperature, IFN-γ level revealed no significant differences; whereas, TNF-α level was significantly lower (p<0.05) in group (d) than in groups (a-c). Besides, concentration of albumin and total protein were significantly higher (p<0.01) in (d) than that in group (a). The levels of magnesium and calcium also echoed similar results (p<0.05). But G-GTP was significantly lower (p<0.05) in group (d) compared with other groups. Therefore, considering the distance between the farm and the laboratory, it is necessary to minimize the effect of temperature and storage time on serum cytokine and biochemical concentration.