With the different experimental parameters, detection of cholesterol in blood sample has considered incredibly significant since its enhancement is related with diabetes, heart diseases, nephrosis, and obstructive jaundice, whereas PiB reduced level of cholesterol is due to malabsorption wasting syndrome, hypothyroidism, and anemia etc. Among the various detection techniques of cholesterol, voltammogramic biosensing 5α-Androstane-3α,17β-diol method has been recently developed as an extremely significant technique. Development of a cholesterol biosensor, immobilization of an enzyme onto selfassembled monolayer fabricated micro-device or bio-chip is usually the primary step in the fabrication of selected biosensor. The selection of an immobilization method is essential for the performance of a biosensor and the future development for fabrication in biosensor design will inevitable focus upon the equipment of innovative devices or chips that recommend assures to resolve the bio-compatibility and bio-fouling problems. Generally, enzymes are biological catalysts that promote the transformation of chemical species in living systems. These biological molecules, consisting of thousands of atoms in precise arrangements, are able to catalyze the multitude of different chemical reactions occurring in biological living cells. Cholesterol enzymes can catalyze reactions in different states: as individual molecules in solution, in aggregates with other entities, and as attached to fabricated surfaces. The attached-or ����immobilized����- state has been of particular interest to those wishing to exploit selective enzymes for practical purposes. The term ����immobilized ChOx enzymes���� refers to ����ChOx enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, and which can be used repeatedly and continuously.���� As a consequence of ChOx enzyme immobilization, some properties of the enzyme molecule, such as its catalytic activity, stability, become altered with respect to those of its soluble counterpart. This modification of the properties may be caused either by changes in the intrinsic activity of the immobilized enzyme or by the fact that the interaction between the immobilized selective enzyme and the substrate takes place in a microenvironment that is different from the bulk solution. The observed changes in the catalytic properties upon ChOx immobilization may also result from changes in the threedimensional conformation of the protein aggravated by the binding of the selective enzyme to the matrix.