Herein,　we　present　the　development　of　an　ultra-sensitive　immobilization-free　homogeneous　electrochemiluminescence　(ECL)　biosensor,　leveraging　the　electrostatic　repulsion　between　a　negatively　charged　indium　tin　oxide　(ITO)　electrode　and　DNA　and　exonuclease　I　(Exo　I)-powered　signal　amplification,　to　achieve　highly　efficient　detection　of　thrombin.　Specifically,　the　aptamer　and　the　complementary　DNA　engage　in　the　formation　of　a　double-stranded　DNA　(dsDNA)　complex.　This　negatively　charged　dsDNA　structure　subsequently　associates　with　a　positively　charged　ECL　indicator,　namely　ruthenium　phenanthroline　(Ru(phen)32+),　resulting　in　the　generation　of　the　dsDNA-Ru(phen)32+　ECL　probe.　The　negatively　charged　dsDNA-Ru(phen)32+　experiences　electrostatic　repulsion　from　the　negatively　charged　ITO　electrode,　resulting　in　a　low　ECL　signal.　Nonetheless,　upon　the　addition　of　thrombin,　the　aptamer　preferentially　binds　to　thrombin,　triggering　the　releases　of　the　embedded　Ru　(phen)32+　facilitated　by　Exo　I　and　hence　resulting　in　a　robust　and　enhanced　ECL　signal.　The　amplified　ECL　signal　is　linearly　correlated　with　the　logarithm　of　thrombin　concentration　within　a　detection　range　spanning　from　10　fmol/mL　to　50　pmol/mL,　with　a　remarkable　detection　limit　of　3.21　fmol/mL.　This　strategy　eliminates　the　need　for　cumbersome　labeling　steps,　avoids　the　electrode　modification　process,　overcoming　the　low　immobilization　efficiency　of　aptamers　and　poor　signal　transduction　of　indicators　labeled　at　the　end　of　DNA.