Fluorescence Lifetime (FLT) of intrinsic fluorophores may alter under the change in metabolic state. In this study, the FLT of rabbit retina was investigated in vivo after laser irradiation using fluorescence lifetime imaging ophthalmoscopy (FLIO). The retina of the Chinchilla bastard rabbits was irradiated with a 514 nm diode laser. FLIO, fundus photography, and optical coherence tomography (OCT) were conducted 30 min and 1 to 3 weeks after treatment. After strong coagulation, the FLT at laser spots was significantly elongated immediately after irradiation, conversely shortened after more than a week. Histological examination showed eosinophilic substance and melanin clumping in subretinal space at the coagulation spots older than one week. The FLT was also elongated right around the coagulation spots, which corresponded to the discontinuous ellipsoid zone (EZ) on OCT. This EZ change was recovered after one week, and the FLT became the same level as the surroundings. In addition, there was a region around the laser spot where the FLT was temporarily shorter than the surrounding area. When weak pulse energy was applied to selectively destroy only the RPE, a shortening of the FLT was observed immediately around the laser spot within one week after irradiation. FLIO could serve as a tool to evaluate the structural and metabolic response of the retina to laser treatments.