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.　©　2022　The　Authors