Abstract: Sulfamethazine was widely used to prevent or treat bacterial diseases in agricultural production such as livestock breeding and aquaculture. However, irrational use of sulfamethazine would lead to excessive residual amount in animal food, which would harm human health. Therefore, rapid and sensitive detection of sulfamethazine was of great significance for food safety and human health. In this work, a novel electrochemical method for the detection of sulfamethazine was constructed based on CRISPR/Cas12a coupled with spherical nucleic acid of gold nanoparticle core to realize rapid, highly sensitive and specific detection of sulfamethazine. The aptamer sequence of the target sulfamethazine was designed as the activator of CRISPR/Cas12a, and the specific binding of sulfamethazine to its aptamer sequence would inhibit the trans-cleavage activity of CRISPR/Cas12a, so that the substrate probe pre-modified on the surface of the gold electrode could be retained, and then the spherical nucleic acid could be introduced to trigger the hybridization chain reaction, producing significantly amplified electrochemical signal. To obtain the best analytical performance, several key conditions including the incubation time of sulfamethazine with aptamer, the concentration and cleavage time of CRISPR/Cas12a, the concentration and reaction time of hairpin probes had been optimized, and the results are 30 min、120 nmol/L、30 min、1 µmol/L、100 min, respectively. Under the optimal conditions, the proposed electrochemical method exhibits prominent quantitative detection performance with the linear detection range of 0.006 ng/mL~600 ng/mL and the detection limit as low as 6 pg/mL, meanwhile, the method shows good distinguishing ability between target antibiotic and other interfering antibiotics. In addition, the method has been successfully applied to the detection of sulfamethazine in actual food samples with the recoveries ranging from 98.07%~106.90%, showing potential application value for food safety detection.