A polyphenol oxidase (PPO) was immobilized by employing magnetic nanoparticles-zinc oxide/zinc hexacyanoferrate (Fe3O4NP-ZnO/ZnHCF) hybrid film electrodeposited on the surface of Pt electrode. The surface functionalization of Fe3O4NP-ZnO/ZnHCF hybrid film was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM), atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS) before and after immobilization of PPO. The biosensor exhibited optimum response within 4s at pH 7.0 and 35°C and linearity in the range 0.04 to 10000 μM for acetaminophen with a detection limit of 0.04 μM (S/N=3). Accuracy of the proposed sensor was found to be 99%. The use of Fe3O4NP/ZnO/ZnOHCF for construction of amperometric acetaminophen biosensor has resulted into relatively rapid response, higher sensitivity, broad linear range, lower detection limit, good reproducibility and long term stability of this biosensor. This sensing interface provides better avenue for the fabrication of various sensor.