Herein, a strategy was put forward for high-sensitive detection of sulfadimethoxine (SDM) using a label-free electrochemical immunosensor. Silver–graphene oxide (Ag-GO) nanocomposites were applied to fabricate the immunosensor. The prepared silver–graphene oxide (Ag-GO) nanocomposites had good biocompatibility and chemical stability. In addition, GO provided large surface area for the assembly of AgNPs with redox activity, which provided a prerequisite for the preparation of the electrochemical immunosensor. Furthermore, gold nanoparticles (AuNPs) were used to immobilize antibodies (anti-SDM) and amplify electrochemical signal, which could further enhance the sensitivity of detection. Under the optimal conditions, the peak current had a linear relationship with the logarithm of the SDM concentration, and the variation range was 0.333 to 100 ng mL−1 with the detection limit of 4 pg mL−1 (S/N = 3). The designed immunosensor demonstrated the advantages of simple preparation, high sensitivity, good selectivity, and acceptable stability. [ABSTRACT FROM AUTHOR]