Electrochemical nanosensors based on nanoporous gold leaf (NPGL) and molecularly imprinted polymer (MIP) are developed for pharmaceutical analysis by using metronidazole (MNZ) as a model analyte. NPGL, serving as the loading platform for MIP immobilization, possesses large accessible surface area with superb electric conductivity, while electrochemically synthesized MIP thin layer affords selectivity for specific recognition of MNZ molecules. For MNZ determination, the hybrid electrode shows two dynamic linear range of 5 × 10−11 to 1 × 10−9 mol L−1 and 1 × 10−9 to 1.4 × 10−6 mol L−1 with a remarkably low detection limit of 1.8 × 10−11 mol L−1 (S/N = 3). In addition, the sensor exhibits high binding affinity and selectivity towards MNZ with excellent reproducibility and stability. Finally, the reliability of MIP-NPGL for MNZ detection is proved in real fish tissue samples, demonstrating the potential for the proposed electrochemical sensors in monitoring drug and biological samples.