Electrodeposited cobalt oxide (CoOx) nanomaterials are not only used for immobilization of cholesterol oxidase (ChOx) but also as electron transfer mediator for oxidation of H2O2 generated in the enzymatic reaction. Voltammetry and flow injection analysis (FIA) were used for determination of cholesterol. FIA determination of cholesterol with biosensors yielded a calibration curve with the following characteristics: linear range up to 50 mM, sensitivity of 43.5 nA mM1 cm2 and detection limit of 4.2 mM. The apparent Michaelis-Menten constant and the response time of the biosensor are 0.49 mM and 15 s, respectively. This biosensor also exhibits good stability, reproducibility and long life time.