An improved amperometric L-lactate biosensor was constructed based on covalent immobilization of lactate oxidase (LOx) from Pediococcus species onto carboxylated multiwalled carbon nanotubes (cMWCNT)/copper nanoparticles (CuNPs)/polyaniline (PANI) hybrid film electrodeposited on the surface of a pencil graphite electrode (PGE). The enzyme electrode was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS), while CuNPs synthesized by chemical reduction method, were characterized by transmission electron microscopy (TEM), UV spectrascopy and X-ray diffraction (XRD). The biosensor showed maximum response within 5 s at pH 8.0 in 0.05 M sodium phosphate buffer and 37 degrees C, when operated at 20 mV s(-1). The biosensor had a detection limit of 0.25 mu M with a wide working range between 1 mu M-2500 mu M. The biosensor was employed for measurement of L-lactic acid level in plasma of apparently healthy and diseased persons. Analytical recovery of added lactic acid in plasma was 95.5%. Within-and between-batch coefficients of variations were 6.24% and 4.19% respectively. There was a good correlation (R-2 = 0.97) between plasma lactate values as measured by standard enzymatic spectrophotometric method and the present biosensor. The working enzyme electrode was used 180 times over a period of 140 days, when stored at 4 degrees C. (C) 2016 Published by Elsevier Inc.