Ractopamine (RAC) is universally known for improving lean meat percentage in livestock and thus is widely introduced as a feed additive. However, it is difficult to eliminate the RAC residue in animal tissues from the biological system and will inevitably harm human health. Hence, detecting RAC molecules in biological samples is extremely significant. Herein, a novel strategy of supramolecular recognition-enhanced electrochemical sensing is presented. This platform was constructed by coupling β-cyclodextrin (β-CD) with palladium nanoparticles (Pd NPs)-functionalized three-dimensional reduced graphene oxide (3D-rGO) to form a nanocomposite (3D-rGO/Pd/β-CD), which was further used to modify a glassy carbon electrode (GCE) for RAC detection. Benefiting from the attractive electrical conductivity and catalytic activity of 3D-rGO/Pd, as well as the unique small-molecule-recognition ability of β-CD demonstrated by 1H NMR spectrum, which revealed the 1 : 2 binding mode of RAC with β-CD, increased peak current signals of RAC were observed in the cyclic voltammetry (CV) test. Under optimized conditions, the wide linear concentration range spanned 1-95 μM, along with a relatively low detection limit of 0.12 μM (S/N = 3), as evidenced by the differential pulse voltammetry (DPV) approach. The platform also exhibited satisfactory stability and fine reproducibility, as well as high selectivity and good anti-interference capability. Moreover, this as-obtained sensor was efficiently applied in pork samples with a high recovery rate (96.44-103.99%), which provides a promising view of its electrochemical biosensing ability in practical applications.