The current spore detection methods rely on cultural techniques, having limitations of time, efficiency, and sensitivity. Spore coat contains calcium dipicolinic acid (CaDPA) as a major constituent, which can serve as a biomarker for bacterial endospores. We report a rapid and sensitive technique for detection of bacterial endospores by using ratiometric fluorescence-based sensors. This method is based on the detection of CaDPA that enhances luminescence of lanthanide ion, when complexed with a semiconducting polymer. A CaDPA standard curve was generated at excitation-emission wavelength of λ₂₈₄-λ₅₂₈ by using Synergy 2 fluorescence spectrophotometer. Intensity was recorded after chelating semiconducting fluorescent polyfluorene (PFO) dots with terbium ions, sensitized by different volumes of CaDPA (0.1 μM). All trials were conducted in the replicates of 3 and mean ± SE were calculated. The standard curve so generated showed a linear relationship (R² = 0.98) in experimental concentration range of 2.5 to 25 nM of CaDPA, with corresponding intensity (a.u.) of 545 to 2130. Endospores of an aerobic spore former, Bacillus licheniformis ATCC 14580, were produced at 37°C for 15 d, on Brain Heart Infusion agar. The efficiency of sporulation was evaluated by spore staining and plating techniques. Total CaDPA content in spores was estimated after suspending reducing concentrations of spores (logs 9.0 through 1.0 cfu/mL, at 1-log intervals) in HPLC-grade water. For higher spore spiking levels such as 9.2 ± 0.03, 8.4 ± 0.05, 7.1 ± 0.13 and 6.3 ± 0.02 logs, the mean CaDPA content values, observed from the standard curve, were 9.4, 7.2, 6.2 and 5.3 nM, whereas, for lower levels of 4.2 ± 0.05, 3.1 ± 0.04, 2.0 ± 0.11, and 1.36 ± 0.09 logs, we observed 3.8, 3.3, 2.2 and 1.3 nM mean CaDPA content. Our results indicated a linear relationship of the CaDPA content of endospores with that of the endospore counts, and the standard curve of CaDPA concentration. This study provides a proof of concept for a potential application of this technique to rapidly detect bacterial endospores in dairy and food industry. Further studies are in progress in our laboratory to standardize this technique for dairy product matrices such as cheese, whey proteins, and powders.