Cholesterol oxidase (ChOx) has been covalently linked to Langmuir-Blodgett (LB) monolayers of polyaniline (PANI)-stearic acid (SA) prepared onto indium-tin-oxide (ITO) coated glass plates via glutaraldehyde (Glu) chemistry. These ChOx/Glu/PANI-SA LB film/ITO electrodes have been characterized by FT-IR, cyclic voltammetry, and scanning electron microscopy, respectively. The results of response measurements carried out on these bioelectrodes using linear sweep voltammetry (LSV) reveal linearity from 25 to 400 mg/dL of cholesterol concentration with sensitivity of 88.9 nA mg(-1) dL. The linear regression analysis of bioelectrode reveals standard deviation and correlation coefficient of 0.737 microA and 0.9988, respectively. The low value of the Michaelis-Menten constant of these bioelectrodes obtained as 1.21 mM for the immobilized enzyme indicates increased interaction between ChOx and cholesterol in the PANI-SA LB film.