Disulfide bonds play an important role in thiol‐based redox regulation. However, owing to the lack of analytical tools, little is known about how local O2 mediates the reversible thiol/disulfide cycle under protein confinement. In this study, a protein‐nanopore inside a glove box is used to control local O2 for single‐molecule reaction, as well as a single‐molecule sensor for real‐time monitoring of the reversible thiol/disulfide cycle. The results demonstrate that the local O2 molecules in protein nanopores could facilitate the redox cycle of disulfide formation and cleavage by promoting a higher fraction of effective reactant collisions owing to nanoconfinement. Further kinetic calculations indicate that the negatively charged residues near reactive sites facilitate proton‐involved oxygen‐induced disulfide cleavage under protein confinement. The unexpectedly strong oxidation ability of confined local O2 may play an essential role in cellular redox signaling and enzyme reactions. The reversible thiol/disulfide cycle under protein confinement was monitored in real‐time using a mutant aerolysin nanopore. Experiments combined with kinetic calculations revealed that confined local O2 can lead to proton‐involved, oxygen‐induced disulfide bond cleavage. The negatively charged neighboring microenvironment facilitates disulfide bond cleavage, which plays a vital role in redox signaling and reactions.