Recently, photothermal therapy (PTT) has been proved to have great potential in tumor therapy. In the last several years, MoS2, as one novel member of nanomaterials, has been applied into PTT due to its excellent photothermal conversion e±cacy. In this work, we applied fluorescence lifetime imaging microscopy (FLIM) techniques into monitoring the PPTtriggered cell death under MoS2 nanosheet treatment. Two types of MoS2 nanosheets (single layer nanosheets and few layer nanosheets) were obtained, both of which exhibited presentable photothermal conversion e±cacy, leang to high cell death rates of 4T1 cells (mouse breast cancer cells) under PTT. Next, live cell images of 4T1 cells were obtained via directly labeling the mitochondria with Rodamine123, which were then continuously observed with FLIM technique. FLIM data showed that the fluorescence lifetimes of mitochondria targeting dye in cells treated with each type of MoS2 nanosheets significantly increased during PTT treatment. By contrast, the fluorescence lifetime of the same dye in control cells (without nanomaterials) remained constant after laser irradiation. These findings suggest that FLIM can be of great value in monitoring cell death process during PTT of cancer cells, which could provide dynamic data of the cellular microenvironment at single cell level in multiple biomedical applications.