The determination of live and dead bacteria is of considerable significance for preventing health care-associated infection in hospitals, field clinics, and other areas. In this study, the viable (live) and nonviable (dead) bacteria in a sample were determined by means of their fluorescence spectra and principal component analysis (PCA). Data obtained in this study show that it is possible to identify bacteria strains and determine the live/dead ratio after UV light inactivation and antibiotic treatment, in situ, within minutes. In addition, synchronous fluorescence scans enable the identification of bacterial components such as tryptophan, tyrosine, and DNA. Compared with the time-consuming plating and culturing methods, this study renders a means for rapid detection and determination of live and dead bacteria.