Efficient　energy　donor-acceptor　pairing　and　distance　control　are　critical　in　resonance　energy　transfer-based　electrochemiluminescence　(ECL-RET)　biosensor.　In　this　work,　a　novel　ECL-RET　system　with　Ru(phen)32+　as　ECL　energy　donor　and　Au　nanocages　as　energy　acceptor　was　constructed　based　on　the　characteristic　that　Ru(phen)32+　can　be　stably　embedded　into　double-stranded　DNA.　On　this　basis,　a　homogeneous　ECL　biosensor　based　on　target　activated　CRISPR-Cas12a　system　and　driving　ECL-RET　effect　was　developed.　The　ECL　biosensor　has　been　successfully　applied　to　the　detection　of　circulating　tumor　DNA　(ctDNA)　and　demonstrated　efficient　recognition　of　L858R　mutation　in　ctDNA　of　non-small　cell　lung　cancer　patients.　The　presented　ECL-RET　biosensor　achieves　a　wide　linear　detection　range　from　10　fM　to　1　nM　and　an　excellent　detection　limit　as　low　as　3.0　fM　for　ctDNA　due　to　its　combination　of　the　highly　efficient　and　specific　cleavage　of　the　CRISPR-Cas12a　system.　©　2025　Elsevier　B.V.