Visualization of glutathione (GSH) enables us to understand GSH-related pathophysiological processes in living subjects. Currently, in vivo visualization methods of GSH are based on visible or first near-infrared (NIR-I) window fluorescence (FL) probes, which possess limitations due to their low tissue penetration depth and strong tissue autofluorescence. Herein, we developed a GSH-activatable second near-infrared (NIR-II) window FL probe (denoted as LET-7) for highly sensitive and selective visualization of GSH in vivo. LET-7, composed of an anionic polymethylcyanide skeleton linked with a FL quenching group of 3,5-bis(trifluoromethyl)benzenethiol, can be specifically activated by GSH, thus triggering a significant NIR-II FL emission enhancement with excellent photostability, which enables us to efficiently distinguish GSH from closely related low-molecular-weight biothiols. The limit of detection of LET-7 for GSH was determined to be as low as 85 nM. Most intriguingly, the in vivo studies demonstrated that LET-7 showed high sensitivity and good selectivity toward GSH. Therefore, our study provides a solution to design activatable NIR-II FL probes for in vivo imaging of GSH and other disease-related biomarkers.