A real-time method employing a portable peptide-containing potentiometric biosensor, can directly detect and/or quantify bacterial spores. Two peptides for specific recognition of B. subtilis and B. anthracis Sterne may be immobilized by a polysiloxane monolayer immobilization (PMI) technique. The sensors translate the biological recognition event into a potential change by detecting, for example, B. subtilis spores in a concentration range of 0.08-7.3×104 CFU/ml. The sensing method exhibited highly selective recognition properties towards Bacillus subtilis spores over other kinds of spores. The selectivity coefficients of the sensors for other kinds of spores are in the range of 0-1.0×10−5. The biosensor method not only has the specificity to distinguish Bacillus subtilis spores in a mixture of B. subtilis and B. thuringiensis (thur.) Kurstaki spores, but also can discriminate between live and dead B. subtilis spores. Furthermore, the sensing method can distinguish a Bacillus subtilis 1A700 from other B. subtilis strain. Assay time may be as low as about 5 minutes for a single test. Rapid identification of B. anthracis Sterne and B. anthracis ΔAmes was also provided.