In this study, electrochemical molecularly imprinted sensors were fabricated and used for the determination of ascorbic acid (AA). Nanofiber membranes of cellulose acetate (CA)/multi-walled carbon nanotubes (MWCNTs)/polyvinylpyrrolidone (PVP) (CA/MWCNTs/PVP) were prepared by electrospinning technique. After being transferred to a glass carbon electrode (GC), the nanofiber interface was further polymerized with pyrrole through electrochemical cyclic voltammetry (CV) technique. Meanwhile, target molecules (such as AA) were embedded into the polypyrrole through the hydrogen bond. The effects of monomer concentration (pyrrole), the number of scan cycles and scan rates of polymerization were optimized. Differential pulse voltammetry (DPV) tests indicated that the oxidation current of AA (the selected target) were higher than that of the structural analogues, which illustrated the selective recognition of AA by molecularly imprinted sensors. Simultaneously, the molecularly imprinted sensors had larger oxidation current of AA than non-imprinted sensors in the processes of rebinding. The electrochemical measurements showed that the molecularly imprinted sensors demonstrated good identification behavior for the detection of AA with a linear range of 10.0 - 1000 μM, a low detection limit down to 3 μM (S/N = 3), and a recovery rate range from 94.0 to 108.8%. Therefore, the electrochemical molecularly imprinted sensors can be used for the recognition and detection of AA without any time-consuming elution. The method presented here demonstrates the great potential for electrospun nanofibers and MWCNTs to construct electrochemical sensors.