Highly sensitive and specific detection and monitoring of trace norepinephrine (NE) in biological fluids and neuronal cell lines is essential for the investigation of pathogenesis of certain neurological diseases. Herein, we constructed a novel electrochemical sensor for real-time monitoring of NE released by PC12 cells based on glassy carbon electrode (GCE) modified with honeycomb-like nickel oxide (NiO)-reduced graphene oxide (RGO) nanocomposite. The synthesized NiO, RGO and the NiO-RGO nanocomposite were characterized using X-ray diffraction spectrogram (XRD), Raman spectroscopy and scanning electron microscopy (SEM). The porous three-dimensional honeycomb-like structure of NiO and high charge transfer kinetics of RGO endowed the nanocomposite with excellent electrocatalytic activity, large surface area and good conductivity. The developed sensor exhibited superior sensitivity and specificity towards NE in a wide linear range from 20 nM to 14 μM and 14 μM–80 μM, with a low detection limit of 5 nM. The performances of the sensor in terms of excellent biocompatibility and high sensitivity allow it to be successfully employed in the tracking of NE release from PC12 cells under the stimulation of K+, providing an effective strategy for the real-time monitoring of cellular NE.