Monitoring and early warning of spores germination is of great significance in avoiding their potential pathogenicity. Thus, effective monitoring of markers during spore germination is of great value. A ratio-dependent fluorescent probe based on in situ incorporation of fluorophores in a metal-organic framework (MOF) was designed to monitor a main component of bacterial spores, 2,6-pyridinedicarboxylic acid (DPA), with high sensitivity and specificity. The fluorescence of CdS quantum dots loaded on zeolitic imidazolate framework-8 (ZIF-8) nanocrystals is initially quenched by europium ions. The europium ions, however, can be seized by DPA, leading to restoring the fluorescence of quantum dots. Simultaneously, the fluorescence of another dye molecule, rhodamine 6G, loaded on the ZIF-8 is not affected by DPA and can serve as a stable internal fluorescence reference signal. On this basis, a ratio-dependent fluorescence method for rapid detection of DPA was established. The linear calibration ranged from 0.1 to 150 μM with a detection limit of 67 nM, which is much lower than the amount of DPA (60 μM) released by the contagious number of spores needed to cause anthrax. This analysis platform exhibits good anti-interference ability for monitoring spore germination. The practicable application of the method was verified by monitoring and imaging the release of DPA in the course of spore germination.