A rapid and simple method for the amperometric determination of glucose using a nanocomposite film of nickel oxyhydroxide and multi-walled carbon nanotube (MWCNTs) was evaluated. The NiHCF)/MWCNT electrode film was fabricated using the liquid–liquid interface method, and it was used as a precursor for the electrochemical synthesis of nickel oxy-hydroxy (Ni(OH)2/NiOOH/MWCNT). The interaction between nickel oxy-hydroxy and the MWCNTs provided a film that is stable over the electrode surface, with high surface area and excellent conductivity. The nanocomposite presented an excellent electrocatalytic activity for the oxidation of glucose in an alkaline medium. The sensitivity of the sensor was found to be 0.0561 μA μmol L−1, and a linear range from 0.1 to 150 μmol L−1was obtained, with a good limit of detection (0.030 μmol L−1). The electrode exhibits a fast response (150 injections h−1) and a sensitive catalytic performance, which may be due to the high conductivity of MWCNT and the increased active surface area of the electrode. Additionally, a minimal difference in the slopes for ascending (0.0561 µA µmol L−1) and descending (0.0531 µA µmol L−1) was observed. Moreover, the sensor was applied to the detection of glucose in artificial plasma blood samples, achieving values of 89 to 98% of recovery.