Enzymes are well known as specialized globular proteins, which catalyses the biochemical reactions without being consumed in the reaction. These enzymes are synthesized in living cells. They function both in vivo and in vitro. When these enzymes are dissolved in buffer, their diameter is around 10 nm. However, when these molecules are made aggregated by desolvation method using ethanol in the nanoscale i.e., 10-200 nm, they are called enzyme nanoparticles (ENPs). These ENPs exhibit their unique optical, electrical, electronic, thermal, chemical and catalytic (ability to facilitate electron transfer) properties beside increased surface area. ENPs are glutaraldehyde cross linked and functionalised by treating with reagents like cysteine/ cysteamine dihydrochloride. Thus, these ENPs are immobilized covalently onto the polycrystalline Au electrode through Au-S- bond. This immobilized ENPs technology has opened a new area for enzyme sensors, biomedical devices and diagnostics to improve their analytic performance. Firstly, ENPs of horse radish peroxidase (HRP) with a diameter of 100 nm (consisting 1000 cross linked enzyme molecules, each of 6 nm in diameter) were prepared and characterized by UV spectro-scopy [1]. These were white in colour and showed absorption peak at 280 nm like free/ natve enzyme molecules. Thereafter, ENPs of few enzymes such as glucose oxidase (GOx), cholesterol oxidase (ChOx), uricase and cholesterol esterase (ChE) were prepared, which measured 117, 100-200, 100 and 35-57 nm in diameter respectively. These were characterized by UV and FTIR spectroscopy and employed in construction of improved amperometric biosensors for measurement of glucose, total cholesterol and uric acid in biological samples, respectively [2-6].