Identification and quantification of plant flavonoids are critical to pharmacokinetic study and pharmaceutical quality control due to their distinct pharmacological functions. Here we report on a novel plant flavonoid electrochemical sensor for sensitive and selective detection of dihydromyricetin (DMY) based on double- layered membranes consisting of gold nanoparticles (Au) anchored on reduced graphene oxide (rGO) and molecularly imprinted polymers (MIPs) modified glassy carbon electrode (GCE). Both rGO-Au and MIPs membranes were directly formed on GCE via in-situ electrochemical reduction and polymerization processes step by step. The compositions, morphologies, and electrochemical properties of membranes were investigated with X-ray powder diffractometry (XRD), Fourier transform infrared spectrum (FTIR), Field emission scanning electron microscopy (FESEM) combined with various electrochemical methods. The fabricated electrochemical sensor labeled as GCE│rGO-Au/MIPs exhibited excellent performance in determining of DMY under optimal experimental conditions. A wide linear detection range (LDR) ranges from 2.0×10-8 to 1.0×10-4 M together with a low limit of detection (LOD) of 1.2×10-8 M (S/N = 3) were achieved. Moreover, the electrochemical sensor was employed to determine DMY in real samples with satisfactory results.