Endopeptidase activity of anthrax lethal factor (aLF) prevents the destroy of anthracis spore intracellularly by host macrophages, meanwhile disables the signaling pathways extracellularly that leads to host lethality. Hence, inhibitory of this activity is expected to be an alternative option to cure anthrax infection. Herein, we fabricated a nanopore platform via transmembrane pore construction in vitro, which allows precise mimics, monitoring of intercellular proteinic transport and enables the quantitative detection of aLF endopeptidase activity towards MAPKK signaling protein at single molecule level. Next, we inhibited the aLF activity via screening approaches of protein-metal ion acquisition and other condition controlment (proton/hydroxide strength, adapted temperature, ionizing irradiation), which were identified by nanopore electrokinetic study. Upon the results, we found that Ca 2+ , Mg 2+ , Mn 2+ , Ni 2+ collaborating with Zn 2+ promote aLF activity efficiently. In contrary, Cd 2+ , Co 2+ , Cu 2+ have great inhibitory effect. Result further revealed that, the speed of aLF endopeptidase activity with different ions functions as the nanopore signal frequency in linear manner, which enables evident distinction of those divalent ions using this proteinase assay. We also found the higher strength of the proton or hydroxide, the higher the inhibitory to aLF activity. Besides, adapted temperature and ?-ray also play integral roles in inhibiting this activity. Our results lay experimental basis for accurate detection of aLF activity, meanwhile provide new direction to screening novel stimuli-responsive inhibitors specific to aLF.