An imprinted electrochemical sensor with specific recognition ability for L-phenylalaine was synthesized with multi layer modified zinc oxide nanofilms(ZnO-NFs),multi walled carbon nanotubes(MWNTs),copper nanoparticles(Cu-NPs) and sol-gel molecularly imprinted polymer(MIP) on the surface of carbon electrode in succession.The morphologies of the modified sensors were investigated by scanning electron microscope(SEM).The electrochemical performance and the optimal conditions for detection of L-phenylalaine were investigated by cyclic voltammetry(CV),differential pulse voltammetry(DPV),amperometric I-t curve and linear sweep voltammetry(LSV).When the scanning rate was 100 mV/s,operating voltage was 0.15 V and pH was 5.5,the calibration curve was linear over the L-phenylalaine concentration range of 5.0×10-8-2.0×10-5 mol/L with a limit of detection of 3.9×10-9 mol/L.This sensor was successfully employed to detect L-phenylalaine in human serum sample with recoveries of 98%-101%.