An electrochemical biosensor fabricated by immobilization of cholesterol oxidase (ChOx) in a polyaniline (PAN)/prussian blue (PB) conductive layer of glassy carbon electrode has been prepared, based on the detection of hydrogen peroxide produced by ChOx at -0.05 V. The properties of the biosensor were investigated and the measurement conditions for cholesterol were optimized. A linear relationship between electrochemical signal and cholesterol concentration in a range of 1×10 -6-8×10 -5 mol/L was observed. It is one of the most sensitive sensors for cholesterol determination, since a low detection limit of 1.8×10 -7 mol/L was found. Good properties of the biosensor were attributed to high activity of ChOx and effective electro-catalysis of PB modifier in the composite layer on electrode surface. An electrochemical biosensor fabricated by immobilization of cholesterol oxidase (ChOx) in a polyaniline (PAN) / prussian blue (PB) conductive layer of glassy carbon electrode has been prepared, based on the detection of hydrogen peroxide produced by ChOx at -0.05 V. The properties of the biosensor were investigated and the measurement conditions for cholesterol were optimized. A linear relationship between electrochemical signal and cholesterol concentration in a range of 1 × 10 -6-8 × 10 -5 mol / L was observed. It is one of the Most sensitive sensors for cholesterol determination, since a low detection limit of 1.8 × 10 -7 mol / L was found. Good properties of the biosensor were attributed to high activity of ChOx and effective electro-catalysis of PB modifier in the composite layer on electrode surface.