The design, construction, and operation of a hand-held synchronously scanned, excitation-emission, double monochromator spectrometer is described. Data show that it is possible to record and display within minutes the fluorescence spectra and ratio of live/dead bacteria in situ. Excitation emission matrix contour plots display clearly bacteria fluorescence spectra before and after UV inactivation, respectively. The separation of the fluorescence band maxima of molecular components, such as tryptophan, tyrosine, and DNA, may be distinguished in the diffused fluorescence spectra of bacteria and mixtures.