A homogeneous assay of the protective antigen in anthrax toxin is reported using two new PA-specific aptamers for selective and sensitive detection, based on reduction in the fluorescence emission according to the formation of the aptamer-PA ternary complex. PA at 1 nM was readily detected using OliGreen as a fluorophore in HEPES buffer. We also demonstrated that the PA detection could be performed in blood serum. The binding interaction between the aptamer and PA was strong enough to dehybridize double-stranded DNA paired completely with 12 bases at room temperature. Moreover, this fluorescence study revealed that the binding sites of the two aptamers were located differently on the PA protein. We believe our approach may lay the groundwork for the real-time detection of PA.