The protein Cu/Zn superoxide dismutase (SOD1) is known to function as a dimer, but its concentration in cells (∼50 μM) and the dimerization constant (K d of 500 μM) results suggest that it exists in a monomer-dimer equilibrium. It is unclear how the oligomeric state of SOD1 changes when cells are initially exposed to high levels of extracellular oxidative stress. To address this problem, we introduced the single-molecule fluorescence anisotropy (smFA) assay to explore SOD1 oligomeric states in live COS7 cells. smFA specifically probes the fluorescence polarization changes caused by molecular rotations where the fast-rotating molecules (either due to smaller hydrodynamic volume or less viscous environments) deteriorate the emission polarization and thus lower the anisotropy. After validating that smFA is effective in distinguishing monomeric and dimeric fluorescence proteins, we overexpressed SOD1 in live COS7 cells and investigated how its oligomeric state changes under basal, 2 h, and 24 h 100 μM H2O2 treatments. We found that treating cells with H2O2 promotes SOD1 dimerization and decreases cellular viscosity in 2 h. Interestingly, prolonged H2O2 treatments show similar results as the basal conditions, indicating that cells return to a steady state similar to the basal state after 24 h, despite the presence of H2O2. Our results demonstrate that SOD1 changes its oligomeric state equilibrium in response to extracellular oxidative stresses. smFA will open new opportunities to explore the relationship between the SOD1 oligomer state and its H2O2-based signaling and transcription regulation roles.