Zearalenone (ZEN), a widespread mycotoxin, can cause great harm to people's health. In order to assay ZEN, an immobilization-free electrochemical sensor has been developed. A multifunctional hairpin DNA has been carefully designed, including three functions: the aptamer for zearalenone (ZEN), primer, and template sequence. This hairpin DNA can anchor on polydopamine nanospheres (PDANSs), which can protect DNA against the digestion of enzymes and prevent the occurrence of strand displacement amplification (SDA). In the presence of ZEN, the hairpin DNA is dissociated from PDANSs due to the interaction between ZEN and the aptamer, and the SDA reaction is initiated with the help of endonuclease and polymerase. During the SDA process, substantial amounts of negatively charged dsDNA are generated. The MB molecules are embedded into the dsDNA grooves to obtain the complex with a negative charge. The confined MB is repelled on the surface of the negatively charged ITO electrode, leading to the decline of the current. This immobilization-free method possesses high sensitivity (LOD of 0.18 pg mL-1) and good selectivity and can be applied to assay ZEN in corn flour.