An electrochemical sensor based on arginine (Arg) functionalized graphene (Arg-G) nanocomposite was fabricated for sensitive detection of acetaminophen. The nanocomposite was characterized by transmission electron microscopy (TEM), Raman spectra etc. The electrochemical behaviors of acetaminophen on Arg-G composite film modified glassy carbon electrode (GCE) were investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The experimental results indicated that the incorporation of arginine and graphene greatly enhanced the electrochemical response of acetaminophen. This fabricated sensor displayed excellent analytical performance for acetaminophen detection over a range from 0.1 to 100 μmol L⁻¹ with a detection limit of 0.05 μmol L^-1 (S/N=3). Moreover, the proposed electrochemical sensor also exhibited good reproducibility and stability, and has been used to detect acetaminophen in tablets and in mouse surem with satisfactory results.