The aggregates of nanoparticles (NPs) are considered better supports for the immobilization of enzymes, as these promote enzyme kinetics, due to their unusual but favorable properties such as larger surface area to volume ratio, high catalytic efficiency of certain immobilized enzymes, non-toxicity of some of the nanoparticle matrices, high stability, strong adsorption of the enzyme of interest by a number of different approaches, and faster electron transportability. Co-immobilization of multiple enzymes required for a multistep reaction cascade on a single support is more efficient than separately immobilizing the corresponding enzymes and mixing them physically, since products of one enzyme could serve as reactants for another. These products can diffuse much more easily between enzymes on the same particle than diffusion from one particle to the next, in the reaction medium. Thus, co-immobilization of enzymes onto NP aggregates is expected to produce faster kinetics than their individual immobilizations on separate matrices. Lipase, glycerol kinase, and glycerol-3-phosphate oxidase are required for lipid analysis in a cascade reaction, and we describe the co-immobilization of these three enzymes on nanocomposites of zinc oxide nanoparticles (ZnONPs)-chitosan (CHIT) and gold nanoparticles-polypyrrole-polyindole carboxylic acid (AuPPy-Pin5COOH) which are electrodeposited on Pt and Au electrodes, respectively. The kinetic properties and analytes used for amperometric determination of TG are fully described for others to practice in a trained laboratory. Cyclic voltammetry, scanning electron microscopy, Fourier transform infra-red spectra, and electrochemical impedance spectra confirmed their covalent co-immobilization onto electrode surfaces through glutaraldehyde coupling on CHIT-ZnONPs and amide bonding on AuPPy/Pin5COOH. The combined activities of co-immobilized enzymes was tested amperometrically, and these composite nanobiocatalysts showed optimum activity within 4-5s, at pH 6.5-7.5 and 35°C, when polarized at a potential between 0.1 and 0.4V. Co-immobilized enzymes showed excellent linearity within 50-700mg/dl of the lipid with detection limit of 20mg/dl for triolein. The half life of co-immobilized enzymes was 7 months, when stored dry at 4°C which is very convenient for practical applications. Co-immobilized biocatalysts measured triglycerides in the sera of apparently healthy persons and persons suffering from hypertriglyceridemia, which is recognized as a leading cause for heart disease. The measurement of serum TG by co-immobilized enzymes was unaffected by the presence of a number of serum substances, tested as potential interferences. Thus, co-immobilization of enzymes onto aggregates of NPs resulted in improved performance for TG analysis.