A novel chitosan-based ratiometric fluorescent probe incorporating an EDTA-Eu3+ complex as the sensing unit and fluorescein dye as the internal standard was designed to detect dipicolinic acid (DPA) as an anthrax bio-marker with high sensitivity and selectivity. The fluorescence intensity of fluorescein dye attached to the chitosan backbone remains constant as an internal reference, while the Eu3+ emission increased linearly upon the consecutive addition of DPA. The selectivity studies were performed by adding different competitive aromatic ligands to the sensing environment and no signifacant fluorescence response was observed. The results demonstrated the superior selectivity of the system to DPA. Overall, this novel chitosan-based ratiometric fluorescent probe enables ratiometric and sensitive DPA detection over nanomolar concentrations (as low as 10 nM) and displays straightforward selectivity over other competitive aromatic ligands.