A core‐shell structure based on polyaniline (PANI) and multiwalled carbon nanotubes (MWCNT) films was electrodeposited on glassy carbon (GC) for the immobilization of acetylcholinesterase (AChE) as enzyme, in developing a biosensor for pesticides. The characterization and advantages of using the hybrid materials were examined by Fourier transform infrared spectroscopy (FTIR), field‐emission gun scanning electron microscopy (FEG‐SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The immobilization of AChE on the surface of the electrode was largely improved due to the unique synergistic effect of PANI and MWCNT. The developed GC/MWCNT/PANI/AChE biosensor was evaluated for determination of carbamate by square wave voltammetry (SWV) and chronoamperometry. SWV allowed carbaryl to be determined in a linear range between 9.9 and 49.6 nmol L−1, with a detection limit of 4.6 nmol L−1 and using chronoamperometry the analytical curve was linear in the concentration ranges from 9.9 to 49.6 µmol L−1, with a detection limit of 1.4 µmol L−1.