A　novel　and　label-free　electrochemical　aptasensor　based　on　exonuclease　III　(Exo　III)　and　G-quadruplex　DNAzyme　has　been　first　designed　to　detect　metronidazole　(MNZ)　sensitively　and　selectively　in　this　paper.　Capture　probe　(CP),　helper　probe　(HP)　and　aptamer　(Apt)　hybridize　to　form　double-stranded　DNA　(dsDNA)　structure　with　a　blunt　3′-terminus,　resulting　in　the　guanine　(G)-rich　CP　being　enclosed　in　the　dsDNA,　followed　by　specific　digestion　of　the　3′-terminus　by　Exo　III　to　expose　the　G-rich　sequence.　At　this　time,　CP　can　bind　with　the　added　hemin　to　form　G-quadruplex　DNAzyme　which　catalyzes　the　redox　reaction　of　3,3′,5,5′-tetramethylbenzidine　(TMB)　and　H2O2,　generating　a　strong　current　signal.　However,　in　the　presence　of　MNZ,　the　binding　of　MNZ　and　aptamer　breaks　the　original　blunt　3′-terminus　of　the　dsDNA　structure,　blocking　the　subsequent　series　of　reactions,　and　only　a　weak　current　signal　can　be　observed.　Under　the　optimal　conditions,　the　aptasensor　showed　excellent　sensitivity　and　specificity　for　the　detection　of　MNZ　with　a　wide　linear　range　from　10　pM　to　500　nM　and　a　detection　limit　as　low　as　3.0　pM　(S/N　=　3).　The　aptasensor　constructed　based　on　MNZ　aptamer　can　effectively　avoid　the　interference　of　other　nitro　compounds　and　antibiotics　to　a　certain　extent.　Compared　with　other　specific　recognition　elements,　the　aptasensor　possesses　the　advantages　of　strong　specificity,　simple　preparation　and　low　cost.　Moreover,　the　proposed　aptasensor　has　been　successfully　applied　to　the　detection　of　MNZ　in　tap　water　and　honey　samples.　©　2022　Elsevier　B.V.