The current study focused on the design of an extremely sensitive electrochemical sensor of ascorbic acid based on a mixture of NiAl2O4-NiO nanoparticles that, produced in a single step using the sol–gel method, on an ITO electrode. This new sensing platform is useful for the detection of ascorbic acid with a wide range of concentrations extending from the attomolar to the molar. SEM micrographs show the porous structure of the NiAl2O4-NiO sample, with a high specific surface area, which is beneficial for the catalytic performance of the nanocomposite. An XRD diffractogram confirmed the existence of two phases, NiAl2O4 and NiO, both corresponding to the face-centred cubic crystal structure. The performances of the modified electrode, as a biomolecule, in the detection of ascorbic acid was evaluated electrochemically by cyclic voltammetry and chronoamperometry. The sensor exhibited a sensitive electrocatalytic response at a working potential of E = +0.3 V vs. Ag/Ag Cl, reaching a steady-state current within 30 s after each addition of ascorbic acid solution with a wide dynamic range of concentrations extending from attolevels (10−18 M) to molar (10 mM) and limits of detection and quantification of 1.2 × 10−18 M and 3.96 × 10−18 M, respectively. This detection device was tested for the quantification of ascorbic acid in a 500 mg vitamin C commercialized tablet that was not pre-treated.