In this work, a NiO–CNT nanocomposite and 2-(3,4-dihydroxyphenethyl)isoindoline-1,3-dione (DPID) were used as modifiers for the modification of a carbon paste electrode (DPID/NiO–CNT/CPE) for the electro-catalytic analysis of glutathione (GSH). The electro-catalytic interaction between DPID and GSH on the surface of the DPID/NiO–CNT/CPE was used as a new way for the analysis of GSH. GSH can be detected on the surface of the DPID/NiO–CNT/CPE in the concentration ranges of 0.03–10.0 and 10.0–200.0 μM with a detection limit of 0.015 μM. The DPID/NiO–CNT/CPE facilitated the simultaneous analysis of glutathione (GSH) in the presence of uric acid (UA) and tryptophan (Trp). The DPID/NiO–CNT/CPE showed high performance ability for GSH analysis in real samples.