Fluorescence analysis applied in the study of lactic acid bacteria (LAB) provides a new method and theory to study probiotics and realize the detection and identification of the strains. It is also possible to achieve automation and computerization. In this letter, the differences between the fluorescence spectra of lactobacillus casei-BDI (Lc-BDI) and Streptococcus thermophilus (St) are shown, and the second-order derivative spectra are used to further study the diversity of these two strains. According to the results, with the excitation wavelengths of 285 and 340 nm, there are significant differences between them. The experiment is repeated for 6 times, showing good repetitiveness.