Poly(ethylenedioxythiophene) (PEDOT) has attracted considerable attention as an advanced electrode material for electrochemical sensors and biosensors, due to its unique electrical and physicochemical properties. Here, we demonstrate the facile preparation of a positively-charged and hierarchical micro-structured PEDOT electrochemical interface with enhanced electrode kinetics for the electrooxidation of NADH. Processable PEDOT colloidal microparticles (PEDOT CMs) were synthesised by template-assisted polymerisation and were then utilised as building blocks for the fabrication of hierarchically-structured electrodes with a larger accessible electroactive surface (2.8 times larger than that of the benchmark PEDOT:PSS) and inter-particle space, thus improving electrode kinetics. The intrinsic positive charge of the PEDOT CMs further facilitated the detection of negatively-charged molecules by electrostatic accumulation. Due to the synergistic effect, these hierarchically-structured PEDOT CMs electrodes exhibited improved NADH electrooxidation at lower potentials and enhanced electrocatalytic activity compared to the compact structure of conventional PEDOT:PSS electrodes. The PEDOT CMs electrodes detected NADH over the range of 20-240 μM, with a sensitivity of 0.0156 μA/μM and a limit of detection of 5.3 μM. Moreover, the PEDOT CMs electrode exhibited a larger peak separation from the interferent ascorbic acid, and improved stability. This enhanced analytical performance for NADH provides a sound basis for further work coupling to a range of NAD-dependent dehydrogenases for applications in biosensing, bio-fuel cells and biocatalysis.