The use of fluorescence spectroscopy for plaque detection is a fast and effective way to monitor oral health. At present, there is no uniform specification for the design of the excitation light source of related products for generating fluorescence. To carry out experiments on dental plaque, the fluorescence spectra of three different bacterial species (Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Streptococcus mutans) were measured by hyperspectral imaging microscopy (HIM). Three critical issues were found in the experiments. One issue was the unwanted spectrum generated from a mercury line source; two four-order low-pass filters were evaluated for eliminating the unwanted spectrum and meet the experimental requirements. The second issue was the red fluorescence generated from the microscope slide made of borosilicate glass; this could affect the observation of the red fluorescence from the bacteria; quartz microscope slides were found to reduce the fluorescence intensity by about 2 dB compared with the borosilicate slide. The third issue of photobleaching in the fluorescence of the Porphyromonas gingivalis was studied. This study proposes a method of classifying three bacteria based on the spectral intensity ratios (510/635 and 500/635 nm) under the 405 nm excitation light was proposed in this study. The sensitivity and specificity of the classification were approximately 99% and 99%, respectively.