Traditional DNA-based electrochemiluminescence (ECL) biosensors generally require a laborious and time-consuming process of electrode modification. Although immobilization-free homogeneous electrochemical approaches have been proposed, the problems of low detectable signals from indicators and high background interferences resulting from label-free sensors remain unsolved. Herein, a new type of homogeneous label-free ECL biosensor for aflatoxin B1 (AFB1) is designed based on a double-driven signal-amplifying strategy (combination of entropy-driven and nuclease-driven) and a background reduction by magnetic graphene oxide (M-GO). The target of AFB1 competitively binds the aptamer in the duplex DNA, leading to the release of the catalyst strand which would further bind the Ru(bpy)32+-Probe (Ru-Probe) absorbed in M-GO based on the principle of entropy increase. In the presence of exonuclease III, the Ru-Probe is degraded to release Ru(bpy)32+ for detection, and the catalyst strand is preserved to trigger much more Ru(bpy)32+ released. So the nuclease-driven combined with entropy-driven amplifying strategy greatly enhances the ECL signal. Moreover, the unreacted Ru-Probes adsorbed in M-GO are removed by the magnetic separation, providing the background signal was reduced as far as possible. Under the optimum conditions, the enhanced ECL signal exhibits a linear relationship with the AFB1 concentration in the range of 1 pM–100 nM with a detection limit of 20 fM. The homogeneous ECL biosensor is successfully applied to determine AFB1 in corn samples with high sensitivity and selectivity. By altering the probes employed, this type of novel ECL biosensor is promising for further determination of diverse targets which are trace contaminants in food, environment, and the disease biomarkers. •A double-driven signal-amplifying strategy incorporated with entropy-driven and nuclease-driven was developed.•M-GO was employed as the receiver of label-free probes and reduced background signal by magnetic separation.•The homogeneous ECL biosensor was immobilization-free of DNA probes and highly sensitive.