This paper reports the fabrication of highly-sensitive and selective voltammetric/amperometric cholesterol biosensor based on cholesterol oxidase co-immobilized with alpha-Fe2O3 micro-pine shaped hierarchical structures. The alpha-Fe2O3 micro-pine shaped hierarchical structures were synthesized by facile hydrothermal process in large quantity and characterized in detail using various techniques. The detailed studies demonstrated that the as-synthesized alpha-Fe2O3 hierarchical structures are grown in very high density exhibiting well-crystallinity and demonstrating rhombohedral alpha-Fe2O3 crystal structures. The fabricated cholesterol biosensors based on alpha-Fe2O3 hierarchical structures exhibited a very high and reproducible sensitivity of 78.56 mu A/mMcm(2) and detection limit (based on S/N ratio) of 0.018 mM. The biosensor exhibited a linear dynamic range from 0.1-8.0 mM and correlation coefficient of R=0.9951. A lower value of apparent Michaelis-Menten constant (K-m(app)), of 0.007 mM, exhibited a high affinity between the cholesterol and ChOx immobilized on alpha-Fe2O3 micro-pine shaped hierarchical structures. To the best of our knowledge, this is the first report in which alpha-Fe2O3 micro-pine shaped hierarchical structures are used for the fabrication of highly sensitive and selective cholesterol biosensor. (C) 2014 Elsevier Ltd. All rights reserved.