Acquiring the concentration of bacteria in water quickly and accurately is of great significance for the supervision hygiene and safety of drinking water. We use multi-wavelength transmission spectroscopy to determine the concentration of bacteria in water, and mainly study the influences of different normalizations of the spectra (particle concentration normalization, maximum normalization, integral normalization, and average normalization) on the accuracy of bacterial concentration. According to the spectra interpretation model of Escherichia coli (E. coli) established on Mie scattering theory, we can obtain the bacteria size by the normalized spectra interpretation, and calculate a single bacterium’s reference spectra. Based on the relation curve between the measured spectra and a single bacterium’s reference spectra, we can obtain the bacterial concentration and analyze the accuracy of the bacterial concentration by several normalization procedures. Compared with plate counting, the bacterial concentration calculated by the average normalization has the maximum relative error of 0.92%, the average relative error of 0.70%, and the correlation coefficient of 0.9984. The accuracy and stability of the method are the best. The study can provide the basic data for the quantitative detection and early warning of bacteria in water.