So far it is unclear whether the release of oxygen-evolving complex (OEC) subunits including PsbO, PsbP, and PsbQ proteins is affected by the phosphorylation of photosystem II (PSII) membranes under light stress. In this work, different phosphorylated PSII membranes were obtained from spinach. Phosphorylation partially suppressed the release of PsbO, PsbP, and PsbQ proteins from PSII membranes under light stress. Reactive oxygen species including superoxide anion, hydrogen peroxide and hydroxyl radical, were involved in the release of a small part of PsbO protein, but not in the release of PsbP and PsbQ proteins in the non-phosphorylated and phosphorylated PSII membranes. All of the results suggested that the release of PsbO, PsbP, and PsbQ proteins was partially regulated by phosphorylation in PSII membranes, and the role of reactive oxygen species in the release of OEC subunits in non-phosphorylated PSII membranes was the same as in phosphorylated PSII membranes.