Fully susceptible tuberculosis (TB) develops secondary drug resistance during therapy due to inappropriate dosing of treatment drugs, inadequate treatment, non-adherence to the prescribed regimen or using low quality medication. Therefore, a fast and reliable method for the determination of a patient's metabolic profile for TB drugs is essential for appropriate dosing of these drugs. A nanobiosensor for the determination of the biotransformation of rifampicin (a TB drug) was developed using ethylene glycol bis(succinic acid N-hydroxysuccinimide ester) (EG)-modified cytochrome P450-2E1 (CYP2E1). The EG-CYP2EI enzyme system was electrodeposited on gold electrodes derivatised with polyvinylpyrrolidone/silver nanoparticles/poly(8-anilino-1-naphthalene sulphonic acid) (PVP-AgNPs/PANSA) nanocomposite. The electrochemical impedance spectroscopy (EIS) and transmission electron microscopy (TEM) analyses of the Au/PVP-AgNPs/PANSA/EG-CYP2E1 nanobiosensor system revealed that PVP-AgNPs/PANSA is a highly electroactive nanocomposite whose morphology and properties were suitable for the immobilisation of CYP2E1. The response profile of the nanobiosensor for rifampicin was studied by cyclic voltammetry (CV), differential voltammetry (DPV) and steady state amperometry under aerobic conditions. The dynamic linear range of the nanobiosensor (0.025 - 14 mu M) covers the peak rifampicin serum level value of 0.045 mu M. (C) 2014 Elsevier Ltd. All rights reserved.