Glutathione (GSH) level in cells maintains redox homeostasis, so the detection of intracellular GSH is crucial for many pathological processes. In the present study, we developed a cystamine-functionalized glass nanopore (cG-nanopore) for the specific detection of GSH in single cells through the Michael addition reaction between the amino group of cystamine and polydopamine adhered to the inner wall of the glass nanopore. The cellular GSH will cleave the disulfide bond of cystamine and induce a negative charge increase on the residual surface of the modified nanopore, thus producing a sensitive response on the current-voltage curves. The developed sensing platform showed a good response relationship with the GSH concentration and an excellent selectivity against interfering substances in cells. The variation of GSH content in single HeLa and H8 cells incubated with DOX was then further quantified by this method.