In the present work, a triple amplified biosensor was constructed for ultrasensitive and selective analysis of an acetylcholinesterase inhibitor; rivastigmine (RIV) in human serum. The modified biosensor based on fabrication of pencil graphite electrode (PGE) with lepidocrocite nanoparticles (γ-FeOOH) dispersed in N-chitosan carbon nanosheets (N@CCNS). Pyrrolidinium acid sulfate (PAS) as an ionic liquid was added to amplify the signal of RIV. The physical properties of the modified electrodes were characterized by powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM) and energy dispersive X-ray (EDX) spectroscopy. The cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) were used to evaluate the performance of the modified electrode. Parameters affecting performance of the modified sensor were evaluated such as pH, scan rate, casting volume, concentration of nanocomposite …etc. The modified sensor showed a fast and sensitive electrochemical response to RIV sensing with wide linear range (3.0–90.0 nM), low detection limit (0.99 nM) and excellent anti-interference ability. Moreover, the fabricated sensor was successfully applied for determination of RIV in pharmaceutical tablets and spiked human serum and the obtained results are quite satisfactory.