An electrochemical biosensor based on ZnO nanorods for potentiometric cholesterol determination is proposed. Hexagon-shaped ZnO nanorods were directly grown on a silver wire having a diameter of 250 μm using low temperature aqueous chemical approach that produced ZnO nanorods with a diameter of 125–250 nm and a length of ~ 1 μm. Cholesterol oxidase (ChOx) was immobilized by a physical adsorption method onto ZnO nanorods. The electrochemical response of the ChOx/ZnO/Ag biosensor against a standard reference electrode (Ag/AgCl) was investigated as a logarithmic function of the cholesterol concentration (1 × 10 −6 M to 1 × 10 −2 M) showing good linearity with a sensitivity of 35.2 mV per decade and the stable output signal was attained at around 10 s.