In this work, a nonenzymatic reduced glutathione (GSH) sensor was constructed based on the electrodepoition of Ni–Al layered double hydroxides (Ni–Al LDHs) on multiwall carbon nanotubes (MWCNTs) modified glassy carbon electrode (GCE). The morphologies and compositions of Ni–Al LDHs/MWCNTs nanocomposite were investigated by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). Cyclic voltammetry and amperometry were employed to explore the electrochemical properties and performance of Ni–Al LDHs/MWCNTs nanocomposites in sensing and detection. The results indicated that the response current of GSH oxidation at the Ni–Al LDHs/MWCNTs/GCE was obviously higher than that at the MWCNTs/GCE or the Ni–Al LDHs/GCE. The amperometric current of the sensor is proportional to the concentration of GSH in a linear range of 1.2 to 1630.0 μM with a detection limit of 0.7 μM at a signal-to-noise ratio of 3. In addition, the sensor exhibits easy preparation, low cost, good stability and anti-interference.