Abstract: In this work, AFM1 was chosen as the target, and the aptamer was split to solve the low sensitivity due to unstable structure of the aptamer. The recognition ability and specificity of the split aptamer were evaluated using electrochemical methods, and the binding mechanism was explored through the circular dichroism. The results showed that the split aptamer employing a 1:1 split mode exhibited the best performance in the recognition and specificity for AFM1. The circular dichroism spectrum remained unchanged. The positive peak intensity of the circular dichroism spectrum increased significantly and the negative peak moved and the intensity increased, which demonstrated that the conformation of the aptamer changed. The sensitivity was enhanced by employing split aptamers. Superior recognition elements for the construction of sensors was obtained by the fragmentation of intact aptamers. The investigation provides a new idea to enhance the sensitivity of the detection mehtods based on the apatamer.