The effect of the laser ablation duration of reduced graphene oxide sheets on their optical properties was studied. After 30 min of ablation, the average lateral size of reduced graphene oxide sheets decreases from 347.4 ± 86.5 nm to 98.8 ± 36.0. The sizes of almost all particles are in the range up to 100 nm, which was confirmed by transmission electron microscopy and dynamic light scattering data. The FTIR spectroscopy data showed that after ablation the intensity of the bands associated with O–H, C–OH and C=O vibrations were noticeably decreased. The optical density and the fluorescence intensity of reduced graphene oxide also depend on the ablation time. After ablation, the reduced graphene oxide fluorescence intensity increased 2–3 times. The fluorescence lifetime decreases both for the first (from 1.36 ns to 0.71 ns) and second (from 6.03 to 3.66 ns) components. A broad band was recorded in the long-lived luminescence spectrum. The long-lived luminescence intensity is higher on 80% for the samples after 30 min of ablation compared to the unablated sample. It was assumed that during laser ablation of reduced graphene oxide a change in the ratio between oxidized and sp2- hybridized carbon occurs. This opens up possibilities for controlling the optical properties of reduced graphene oxide.