Display omitted ► The hemin/G-quadruplex simultaneously acts as NADH oxidase and HRP-mimicking DNAzyme. ► The PdNPs-RGs were adopted to further enhance the electrochemical signal. ► The avidin–biotin system was employed to improve the sensitivity of thrombin analysis. ► The prepared aptasensor exhibited low detection limit and wide linear range to TB. In this work, an advanced sandwich-type electrochemical aptasensor for thrombin was proposed by integrating hemin/G-quadruplex with functionalized graphene-Pd nanoparticles composites (PdNPs-RGs). The hemin/G-quadruplex formed by intercalating hemin into thrombin binding aptamer (TBA), firstly acted as a NADH oxidase, assisting the oxidation of NADH to NAD+ accompanying with the generation of H2O2 in the presence of dissolved O2. Subsequently, the hemin/G-quadruplex acted as HRP-mimicking DNAzyme that rapidly bioelectrocatalyze the reduction of the produced H2O2. At the same time, the Pd nanoparticles supported on p-iodoaniline functionalized graphene were also adopted to catalyze the reduction of H2O2. Thus, with the dual catalysis, a dramatically amplified electrochemical signal could be obtained. Besides, the avidin–biotin system for binding aptamer sequences on electrodes not only improved the sensitivity of thrombin analysis but also obtained an acceptable repeatability of the aptasensor. With several factors mentioned above, a wide linear ranged from 0.1pM to 50nM was acquired with a relatively low detection limit of 0.03pM (defined as S/N=3). These excellent performances provided our approach a promising way for ultrasensitive assay in electrochemical aptasensors.