The monitor of pathogenic microorganisms in water is important for public health and disease outbreaks prediction. Recently, the optical detection techniques have drawn much attention due to the advantages of rapid response, security and high sensitivity. In this paper, a fluorescence spectrometer based on 375 nm exciting laser and the microchannel liquid sample flow technology is proposed. The 4×4 narrowband filter array was used as a spectroscopic system for high fluorescence flux, which was coupled to a Silicon Photomultiplier (SiPM) array with single-photon sensitivity. B500 fluorescent microspheres and Escherichia coli were used for performance evaluation of the spectrometer. As a result, it is feasible to use random particle counting method to detect the bacteria concentration level in water even low to a single CFU/mL. In addition, the method was successfully applied to tap water samples. The results suggest that the proposed method is applicable for on-site bacteria detection.