Graphene oxide (GO), the oxidized state of graphene, has a similar chemical structure to graphene, except for the existence of many defects and oxygen-containing functional groups. GO could be photoreduced under light irradiation, and it is crucial to study and understand the changes of chemical structure and photoluminescence during this photoreduction process. Herein, by using single-molecule optical microscopy, the photoreduction of three different GO sheets were monitored in situ. The photoluminescence from highly oxidized GO sheets changed continuously under light irradiation, and many blinking spots showed up on this GO sheet surface. The appearance of these blinking events is highly related to the stacking of the GO sheets. Much less blinking could be observed on stacked GO sheets. It was found that the reduction speed of GO in water is much higher than that in air, and much less blinking could be observed when GO was reduced in water. Moreover, this is also highly related to the chemical structures of the GO sheets used for photoreduction. No blinking could be observed on less oxidized GO sheets. This work provides an effective approach for in situ study of the optical properties of GO sheets, boosting a deeper understanding of GO photoreduction. Moreover, this method could also be extended for in situ studying the optical properties of other materials.