Herein, an electrochemical ascorbic acid (AA) sensor was fabricated based on the MnO2/multi-walled carbon nanotubes (MWNTs-COOH) composite film modified glassy carbon electrode (GCE). The scanning electron microscopy (SEM) was used to characterize the morphologies of MnO2/MWNTs-COOH composite film. The MnO2/MWNTs-COOH/GCE was employed to detect AA by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The experiment test results showed that the proposed electrode exhibited an obvious electrocatalytic ability to the oxidation of AA. The DPV was employed to analyze AA in 0.1 M PBS (pH=7.0), the oxidation peak current of AA displayed a good linear relationship with the logarithm of the concentration of AA in the range of 1.0 × 10−6 - 1.0 × 10−4 mol/L, the detection limit was 2.7 × 10−7 mol/L (S/N = 3). Additionally, the results indicated that the constructed method possessed high selectivity, long-term stability and excellent reproducibility, and successfully used to detect AA in water samples with satisfactory results.