A layer-by-layer (LbL) assembly technique was employed to modify glassy carbon electrodes (GCEs) and screen printed electrodes (SPEs) utilizing multiwalled carbon nanotubes (MWCNTs)/polyelectrolyte binary composites and an enzyme cascade to facilitate efficient electron transfer in sucrose/O 2 biofuel cell. In this study, MWCNTs immobilized invertase (INV) and glucose dehydrogenase (GDH) were alternatively assembled upon polyethyleneimine (PEI) and DNA nanocomposites to construct a bioanode.[Ni(phendion)(phen)]Cl 2 complex and methylene green (MG) were investigated as redox mediators for electrocatalytic oxidation of NADH at reduced overpotentials. The LbL architecture showed advantages for sequential enzymatic reaction that favored the efficient penetration of substrate and products in a cascade system while MWCNTs facilitated the efficient electron transfer from sucrose oxidation and enhancement of the current density. With a GCE bioanode modified with a MG or Ni complex, the biofuel cell produced a higher power density (μW/cm 2 ) with 145.8% and 130.11% enhancement comparing to a SPE bioanode, respectively. Moreover, the Ni complex modified GCEs and SPEs produced power densities (μW/cm 2 ) 93.7% and 107.0% higher compared to MG modified bioanodes, respectively. The maximum current density of 1400 ± 46 μA/cm 2 was obtained with the Ni complex on GCE at an OCP of 604 ± 17 mV with a maximum power density of 405 ± 6 μW/cm 2 . The LbL assembly showed great feasibility as a simple and efficient way to construct controlled MWCNT-multi-enzyme modified electrodes for biofuel cell applications. Biofuel cells convert the chemical energy of biofuels into electric energy via the enzymatically catalyzed oxidation and reduction reactions. One of the most important factors affecting the performance of fuel cell is the fabrication of bioanode which has great influence in generation of electrical power outputs. Therefore developing effective bioanodes remains critical. Till date, several reports have been published on various approaches to assemble enzymes on the anodes in BFCs utilizing carbon based materials.1-4 Amongst those layerby-layer (LbL) assembly technique via electrostatic interactions of oppositely charged species has emerged as a very attractive way to construct multilayer films of polyelectrolytes, biomolecules, 5 and organic materials 6-8 offering advantages in various fields, such as surface functionalization, 9,10 drug delivery, 11,12 and biosensing. [13][14][15] Its application was considered plausible in the development of amperometric biosensors initiating vast research activities on biosensors comprised of LbL organized multilayers. LbL nanostructures decorated with multi-enzymes were proven to be one of the successful strategies to establish high electrical performance, long-term stability and long lifetime in bioelectronics devices.14,16-18 For example, LbL structures consisting of Au nanoparticles (AuNPs), thiol-functionalized polyaniline and glucose oxidase (GOx) were fabricated for glucose biosensing ...