Conducting polymers have attracted considerable attention as potential electrode materials in electrochemical sensors and biosensors. While conducting polymers possess unique electrical and physicochemical properties, they suffer from the drawback of poor processability because of interchain interactions. Here, we introduce the concept of aqueous processable poly(ethylenedioxythiophene) microparticles (PEDOT-MPs) in colloidal dispersion synthesized by CaCO3 template-assisted method. The processable PEDOT-MPs was utilised as building blocks for the fabrication of electrodes with hierarchically-structured interfaces. Compared to commercial PEDOT:PSS electrodes, which have a dense film, the electrodes prepared with the colloidal PEDOT-MPs possess a hierarchically-structured film, which provides a larger accessible active surface and inter-particle space facilitates molecular diffusion. Taking NADH as a model analyte, such novel PEDOT-MPs electrodes detect NADH at a lower operation potential and with enhanced electrocatalytic activity (oxidation peak 1.7 times higher) compared to the commercial PEDOT:PSS electrode. The calculated diffusion coefficient of the hierarchically-structured PEDOT-MPs electrode was 1.61 x 10-6 cm2 s-1, which is ~4 times higher than that of the dense PEDOT:PSS electrode (3.98 x10-7 cm2 s-1). The PEDOT-MPs electrodes detected NADH over the range from 20 to 240 uM, with a sensitivity of 15.61 uA/mM and a limit of detection of 5.3 uM. Moreover, the hierarchically-structured PEDOT-MPs electrode exhibited improved stability (1.3 times higher over 10 measuring cycles) compared to the commercial PEDOT:PSS due to reduced fouling by the oxidised products (NAD+ and dimer), which can be ascribed to the positive surface charges (21.3 ± 4.27 mV) of the PEDOT-MPs. This colloidal PEDOT-MPs show good processability for facile preparation of electrodes, and micro-structured morphology to enhance the analytical performance for NADH biosensing, as well as reduced of NAD+ and dimer fouling. Our developed PEDOT-MPs hierarchically-structured electrodes could potentially coupled with NAD-dependent dehydrogenase enzymes for the development of wide range of biosensors.