In this work, a kind of homogeneous electrochemical aptasensor based on DNA assembly was designed to detect zearalenone (ZEN). The three DNA strands of ZEN aptamer (Apt), complementary strand DNA1, and complementary strand DNA2 are first mixed to form DNA duplex structure, which cannot be cleaved by RecJfExonuclease (RecJfExo) and hinders the electron transfer after addition to the electrode surface. With the existence of ZEN, the combination of ZEN and Apt destroys the duplex structure, and RecJfExo can catalyze the cleavage of Apt to form short DNA strands, which accelerates the electron transfer after addition to the electrode surface. According to the current difference with or without ZEN, the detection purpose is achieved. The linear relationship between Iand LgCZENwas found in the range of 5.0 × 10−6ng/ml to 50 ng/ml, and the detection limit is as low as 0.51 fg/ml. The prepared sensor showed high selectivity, favorable repeatability, and reproducibility and was effectively used for testing the spiked corn flour and beer samples. The three DNA strands of ZEN aptamer, DNA1 and DNA2 are first mixed to form DNA duplex structure, which cannot be cleaved by RecJf Exo and hinders the electron transfer after addition to the electrode surface. With the existence of ZEN, the combination of ZEN and Apt destroys the duplex structure, and RecJf Exo can catalyze the cleavage of Apt to form short DNA strands, which accelerates the electron transfer. Therefore, the detection purpose is achieved.