Cholesterol is one of the main biomarkers for atherosclerosis which can lead to the coronary heart and peripheral arterial diseases. Development of non-enzymatic electrochemical biosensors for the measurements of cholesterol over a wide linear concentration range, with high sensitivity and accuracy is challenging. A novel Cu/Ni bimetal-dispersed carbon nanofiber/polymer nanocomposite (BMCP)-based electrode was developed in this study for the measurements of cholesterol. Dendritic nanofibers of poly methyl orange (PMO), which served as the recognition element for cholesterol, were successfully grown over BMCP, using electro-polymerization. Tested using various electrochemical techniques including chronoamperometry and differential pulse voltammetry, the prepared PMO-BMCP biosensor showed a high sensitivity (226.30 mu A mM(-1) cm(-2)) and low detection limit (0.002 mg dL(-1)) over 0.04-600 mg dL(-1) concentration range, with remarkable linearity (R-2 = 0.999), which were either higher or comparable to most of the cholesterol sensors discussed in literature. The biosensor exhibited good reproducibility and long-time (similar to 8 months) stability of the electrochemical activity. The presence of interfering bioactive agents, namely, glucose, uric acid, creatinine, urea and p-acetamido phenol had negligible effect on the measurement of cholesterol. The proposed material and method described in this study can be used to develop similar non-enzymatic electrochemical biosensors for other bioactive molecules in blood, such as glucose and creatinine.