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Bacterial concentration is an important indicator to measure the degree of water pollution. Realizing rapid and accurate quantification of bacterial concentration in water is of great significance for ensuring water safety and maintaining human health. This paper proposes a method for rapid determination of bacterial concentration by multiwavelength transmission spectroscopy combined with partial least squares regression. Escherichia coli (E. coli) is selected because it is a common indicator microorganism for assessing water pollution status, and it is easy to handle. First, we measure the transmission spectra for E. coli suspensions in the region from 200 to 900 nm and analyze the differences in the spectral characteristics at different concentrations; subsequently, considering that the concentration is affected by the instrument linearity and other factors, the sensitivity, correlation, and detection ability of the spectra at different wavelengths with the change of concentration are analyzed, and the optimal characteristic band is selected according to its wavelength variation characteristics; finally, the determination of E. coli concentrations are completed by using the optimal characteristic band spectra combined with partial least squares regression. We calculate the bacterial concentration, compared with the plate counting, the maximum relative error is 4.500%, the average relative error is 0.677%, respectively, which is less than 5%, and their accuracy and stability are all better than those calculated by the single-wavelength method. This study provides a reference for the rapid and accurate detection of bacterial concentration in water. [ABSTRACT FROM AUTHOR]