Nanoparticles (NPs) of commercial lipase from Candida rugosa, of glycerol kinase (GK) from Cellulomonas species, of glycerol-3-phosphate oxidase (GPO) from Aerococcus viridans were prepared, characterized and co-immobilized onto a pencil graphite (PG) electrode. The morphological and electrochemical characterization of PG electrode was performed by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) before and after co-immobilization of enzyme nanoparticles (ENPs). An improved amperometric triglyceride (TG) biosensor was fabricated using Lipase NPs/GKNPs/GPONPs/PG electrode as the working electrode, Ag/AgCl as the standard electrode and Pt wire as auxiliary electrode. The biosensor showed optimum response within 2.5 s at a pH 7.0 and temperature of 35 degrees C. The biosensor measured current due to electrons generated at 0.1 V against Ag/AgCl, from H-2 O-2, which is produced from triolein by co-immobilized ENPs. A linear relationship was obtained over between a wide triolein concentration range (0.1 mM-45 mM) and current (mA) under optimal conditions. The Lipase NPs/GKNPs/GPONPs/PG electrode showed high sensitivity (1241 +/- 20 mA cm(-2) mM(-1)); a lower detection limit (0.1 nM) and good correlation coeficient (R-2 = 0.99) with a standard enzymic colorimetric method. Analytical recovery of added triolein in serum was 98.01%, within and between batch coefficients of variation (CV) were 0.05% and 0.06% respectively. The biosensor was evaluated and employed for determination of TG in the serum of apparently healthy subject and persons suffering from hypertriglyceridemia. The biosensor lost 20% of its initial activity after its continued uses over a period of 240 days, while being stored at 4 degrees C. (C) 2017 Elsevier Inc. All rights reserved.