＂Cu-DNAzyme　＂　and　＂G4-DNAzyme　＂　were　used　to　develop　a　＂turn-off　＂　dual-DNAzyme　colorimetric　biosensor,　which　could　be　used　to　detect　Cu2+　by　employing　exonuclease　III-mediated　cyclical　assembly　(EMCA).　EMCA　was　based　on　the　cleavage　activity　of　Cu2+　to　transfer　the　linkage　sequences　of　the　substrate　strand　and　enzyme　strand　into　the　transition　sequence.　The　horseradish　peroxidase　(HRP)-mimicking　activity　of　the　G4-DNAzyme　was　lost　after　binding　with　the　complementary　transition　sequence　and　was　hydrolyzed　by　Exo　III.　These　results　demonstrate　that　the　proposed　colorimetric　biosensor　was　an　effective　method　for　ultradetection　of　trace　metals　in　a　high　original　signal　background.　Due　to　the　high　sensitivity　of　the　biosensor,　the　limit　of　detection　(LOD)　of　Cu2+　is　0.16　nM.　This　design　offers　a　general　purpose　platform　that　could　be　applied　for　the　detection　of　any　metal　ion　target　through　adjustment　of　metal-dependent　DNA-cleaving　DNAzymes,　which　is　of　great　significance　for　the　rapid　determination　of　food　safety.