Cholesterol oxidase (ChOx) has been immobilized onto conducting poly[2-methoxy,5-(2′-ethyl-hexyloxy)-1,4-phenylene vinylene] (MEH-PPV)/stearic acid (SA) Langmuir–Blodgett film transferred onto octadecanethiol (ODT) modified gold plate. The ChOx/MEH-PPV/SA LB film bioelectrode exhibits has been characterized by FT-IR, contact angle, and atomic force microscopy. The response of the ChOx/MEH-PPV/SA LB film bioelectrode carried out using differential pulse voltammetry (DPV) studies reveal linearity from 1.29 to 12.91mM of cholesterol concentration and response time as 30s. This ChOx/MEH-PPV/SA bioelectrode exhibits values of correlation coefficient as 0.9939, standard deviation as 0.0029μA and limit of detection as 1.66mM. UV–visible spectrophotometer studies reveal that 5.2×10 −3 U of ChOx are actively working per cm 2 area of ChOx/MEH-PPV/SA LB film bioelectrode and this bioelectrode is thermally stable upto 55°C with reusability of about 60 times.