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The rapid and sensitive visualization of 2,6-dipicolinic acid (DPA, a unique anthrax biomarker) is essential to prevent anthrax disease or biological terrorist attack. In this study, a Eu3+-labeled ethylenediaminetetraacetic acid loaded hyperbranched polyethyleneimine carbon nanodot (hPEI-CD-EDTA-Eu3+) nanoprobe has been proposed for the ratiometric DPA detection. The sensing mechanism is based on the rapid DPA-Eu3+ chelation within 30 s and subsequent enhanced fluorescence emission through the antenna effect. With the introduction of EDTA chelating unit, the resulted fluorescence of Eu3+-complex is greatly enhanced, which endows sensitive DPA perception. By employing hPEI-CD as the internal reference, ratiometric DPA sensing is realized with a good linearity in the concentration range from 1.0 to 100 nM, with a limit of detection of 190 pM (S/N = 3). The specific chelation affinity between Eu3+ and DPA provides satisfying selectivity over other amino acids and ions. Using nanoprobe-loaded polyvinylidene fluoride paper as the analytical device, point-of-care DPA visualization is achieved. Furthermore, the practical application of designed paper device is validated by the visual detection of metabolic DPA-release from Bacillus subtilis spores. Image 1 • EDTA chelation can greatly enhance the fluorescence of Eu3+‒DPA complex. • Eu3+-labeled carbon nanodot fluorescence nanoprobe for sensitive DPA detection was fabricated. • Other metal ions or amino acids had no interference on DPA sensing. • The point-of-care DPA detection was achieved using nanoprobe-loaded PVDF paper device. • The visualization of metabolic DPA-release from Bacillus subtilis spores was realized. [ABSTRACT FROM AUTHOR]