In　this　paper,　a　novel　and　sensitive　electrochemical　aptasensor　for　sulfadimethoxine　(SDM)　detection　has　been　designed　based　on　the　triple　helix　structure/exonuclease　I　(Exo　I)-assisted　double　signal　amplification　strategy.　The　aptamer　probe　(Apt)　hybridizes　with　the　signal　transduction　probe　(STP)　on　the　electrode　to　form　a　rigid　double-stranded　DNA　(dsDNA)　structure,　so　that　the　STP　remains　upright　and　methylene　blue　(MB)　on　the　STP　is　far　away　from　the　electrode　surface,　resulting　in　a　delicate　current　signal.　In　the　presence　of　SDM,　the　SDM　and　Apt　combine　into　a　complex,　leading　to　the　transfer　of　the　Apt　and　the　exposure　of　the　STP.　Meanwhile,　the　added　Exo　I　can　digest　the　Apt　to　realize　the　cyclic　amplification　of　SDM.　After　the　addition　of　the　signal　probe　(SP),　a　triple　helix　structure　between　the　SP　and　STP　is　formed　under　acidic　conditions,　and　MB　on　the　STP　and　SP　collide　with　the　electrode　surface　to　generate　a　strong　electrochemical　signal.　The　proposed　aptasensor　combines　the　features　of　the　triple　helix　structure　and　Exo　I　to　achieve　double　signal　amplification　for　the　sensitive　detection　of　SDM　with　a　wide　linear　range　of　0.05-1000　ng　mL−1　and　a　low　detection　limit　of　0.02　ng　mL−1.　Furthermore,　it　has　been　successfully　used　to　detect　SDM　in　milk　and　lake　water　samples.　©　2024　The　Royal　Society　of　Chemistry.