An electrochemical sensor was developed for detecting zearalenone (ZEN) based on the mimic peptide, which was screened from the library and validated by molecular simulation and electrochemical methods. The library of the mimic peptide was constructed according to the structural analysis, molecular docking, molecular dynamics and amino acid mutation. Then, the enhanced electrical signal was attributed to gold nanoparticles (AuNPs) and reduced carboxylated graphene oxide (rGO-COOH). Under the optimal conditions, the detection limit was 0.91 pg·mL−1 (S/N = 3) with a wide linear range from 0.01 ng·mL−1 to 10 ng·mL−1. In grain samples, a good recovery rate of 84% to 105.3% was achieved, while the rate ranged from 82% to 108.8% in the commercial ELISA kits. Additionally, the electrochemical sensor exhibited the remarkable specificity, excellent stability and better reproducibility (RSD = 1.94%). This sensor has great potential for rapidly detecting ZEN in food.