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.