We report on our investigation of the spatial distribution and autofluorescence lifetime characterization of lipofuscin and oxidized melanin in the retinal pigment epithelium cells of the pig eye using a two-photon excitation fluorescence lifetime imaging microscopy (TPE-FLIM) system, which that is based on a time-correlated single photon counting technique. In particular, we analyzed the difference of autofluorescence lifetimes of these pigment granules in light-induced oxidizing environment. The experimental results showed that the fluorescence lifetime imaging can provide an effective differentiation of multi-component fluorophores, and fluorescence decay can be used to distinguish normal from abnormal fluorescence. TPE-FLIM has the potential to provide a high sensitive imaging instrument for the clinical diagnosis and pathological studies in ophthalmology, and is also of significance to the study of aging mechanism of cells in the fundus.