In this work, a novel manganese dioxide-graphene nanosheets (MnO-GNSs) composite was synthesized by a facile one-step hydrothermal method, in which manganese dioxide (MnO) was fabricated by hydrothermal reduction of KMnO with GNSs. The structure and morphology of MnO-GNSs composite were characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD) analysis and X-ray photoelectron spectroscopy (XPS). A sensitive non-enzymatic electrochemical sensor based on MnO-GNSs composite for the detection of low concentration hydrogen peroxide (HO) was fabricated. The electrochemical properties of MnO-GNSs composite modified glassy carbon electrode (MnO-GNSs/GCE) were investigated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and amperometry. The observations confirmed that the fabricated sensor exhibited high electrocatalytic activity for oxidation of HO owing to the catalytic ability of MnO particles and the conductivity of GNSs. Under the optimum conditions, the calibration curve was linear for the amperometric response versus HO concentration over the range 0.5-350 μM with a low detection limit of 0.19 μM (S/N = 3) and high sensitivity of 422.10 μA mM cm. The determination and quantitative analysis of HO in antiseptic solution on MnO-GNSs/GCE exhibited percent recovery of 96.50%-101.22% with relative standard deviation (RSD) of 1.48%-4.47%. The developed MnO-GNSs/GCE might be a promising platform for the practical detection of HO due to its prominent properties including excellent reproducibility, good anti-interference and repeatability.