The invention provides a preparation method of an electrochemical adapter sensor for detecting fumonisin B1 (FB1). The preparation method comprises the following steps of step one, preparation of an silk-printed carbon electrode (SPCE); step two, preparation of an activated FB1 aptamer solution; step three, preparation of an Au nanoparticles (AuNPs) modified SPCE; step four, preparation of an FB1 aptamer modified AuNPs-SPCE; step five, sealing of spare active sites on the surface of the electrode. According to the preparation method of the electrochemical adapter sensor for detecting the FB1, provided by the invention, the AuNPs are adopted for modifying the surface of the electrode, on one hand, the large amount of AuNPs are loaded on the surface of the electrode, so that a signal amplification function on label-free impedance method detection is obtained; on the other hand, more sulfhydrylated adapters are fixed to the surface of the electrode by utilizing Au-S bonds. An FB1 impedance detection method provided by the invention has the characteristics that the operation is more simple, convenient and flexible, instruments and equipment are simpler, the dosage of a reagent is less, the detection cost is low, and the like.