A novel nanobiocomposite bienzymatic amperometric cholesterol biosensor, coupled with cholesterol oxidase (ChOx) and horseradish peroxidase (HRP), was developed based on the gold-nanoparticle decorated graphene-nanostructured polyaniline nanocomposite (NSPANI-AuNP-GR) film which was electrochemically deposited onto indium-tin-oxide (ITO) electrode from the nanocomposite (NSPANI-AuNP-GR) dispersion, as synthesized by in situ polymerization technique. The gold nanoparticle-decorated graphene-nanostructured polyaniline nanocomposite (NSPANI-AuNP-GR) offers an efficient electron transfer between underlining electrode and enzyme active center. The bienzymatic nanocomposite bioelectrodes ChOx-HRP/NSPANI-AuNP-GR/ITO have exhibited higher sensitivity, linearity, and lower value than monoenzymatic bioelectrode (ChOx/NSPANI-AuNP-GR/ITO). It is inferred that bienzyme-based nanobioelectrodes offer wider linearity (35 to 500？mg/dL), higher sensitivity (0.42？μAmM？1), low km value of 0.01？mM and higher accuracy for testing of blood serum samples than monoenzyme system. Mechanism of the overall biochemical reaction has been proposed to illustrate the enhanced biosensing performance of the bienzyme system. The novelty of the electrode lies on reusability, extended shelf life, and accuracy of testing blood serum samples. 1. Introduction Graphene, one of the most exciting nanostructures of carbon, is a two-dimensional honeycomb crystalline single layer of carbon lattice [1–3]. Recently, it has received enormous interest in various areas of research, such as biosensors, bioelectronics, energy storage and conversion, drug delivery [4–7], molecular resolution sensors [8–10], ultrafast electronic devices, [11], and electromechanical resonators [5], owing to its large specific surface area, extraordinary electrical and thermal conductivities [11, 12], high mechanical stiffness [13], good biocompatibility [14], and low manufacturing cost [15]. The high electrical and thermal conductivities of graphene originate from the extended long-range -conjugation. Out of all conducting polymers, polyaniline (PANI) is one of the promising matrixes for biosensor applications due to its simple and reversible acid/base doping/dedoping chemistry enabling control over properties such as free volume [16], solubility [17], electrical conductivity [18], and optical activity [19]. In recent years, nanostructured polyaniline (NSPANI) has aroused much scientific interest since it combines the properties of low-dimensional organic conductors and high-surface-area materials and offers the possibility
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