Hematocrit (HCT) is actually a calculated value obtained from modern automated hematology analyzers. It is the product of the mean cell volume (MCV) and the red blood cell (RBC) count, both of which are directly measured by the analyzer. Therefore, if there are any inaccuracies in measurement of the MCV or RBC count, the HCT will reflect those inaccuracies. In this study we have demonstrated the feasibility of electrochemical methods for determination of hematocrit level in whole blood samples. The sensitivity of the hematocrit biosensor was 1.01μA/% and the average coefficient of variation was 5.04%, respectively.