A new fluorescent probe(Rhod-Sec) for selenol detection with ultralow background fluorescence have been developed in this paper, which showed a 380-fold off-on fluorescence response, and the nontoxic Rhod-Sec is well suitable for detecting and imaging both exogenous and endogenous selenol in living cells. It also can be applied to visualize the fluctuation of selenol in HepG2 cells through LPS-induced cells oxidation resistance. A new fluorescent probe (Rhod-Sec) for selenol detection with ultralow background fluorescence have been developed in this paper, which showed a 380-fold off-on fluorescence response, and the nontoxic Rhod-Sec is well suitable for detecting and imaging both exogenous and endogenous selenol in living cells. It also can be applied to visualize the fluctuation of selenol in HepG2 cells through LPS-induced cells oxidation resistance.