To research the lethal mechanism of spores stressed by alkali, laser tweezers Raman spectroscopy (LTRS) combined with principal components analysis (PCA) was used to study the physiological process of single spore with alkali stress. The results showed that both spores and germinated spores had tolerance with alkali in a certain range, but the ability of spores was obviously lower than that of spores due to the release of their Ca2+-DPA which plays a key role in spores resistance as well as spores resistance to many stresses; A small amount of Ca2+-DPA of spores was observed to release after alkali stress, however, the behavior of release was different with the normal Ca2+-DPA release behavior induced by L-alanine; The data before and after alkali stress of the spores and g.spores with PCA reflected that alkali mainly injured the membrane of spores, and alkali could be easily enter into the inner structure of spores to damage the structure of protein backbone and injure the nucleic acid of spores. We show that the alkali could result in the small amount of Ca2+-DPA released by destroying the member channel of spores.