A novel sandwich-type electrochemical aptasensor has been fabricated and applied for sensitive and selective detection of antibiotic oxytetracycline (OTC). This sensor was based on graphene-three dimensional nanostructure gold nanocomposite (GR-3D Au) and aptamer-AuNPs-horseradish peroxidase (HRP) nanoprobes as signal amplification. The aptamer and HRP modified Au nanoparticles provide high affinity and ultrasensitive electrochemical probe with excellent specificity for OTC.