Colorimetry camera-based fluorescence microscopy (CCFM) is a single-frame imaging method for observing multiple biological events simultaneously. Compared with the traditional multi-color fluorescence microscopy methods based on sequential excitation or spectral splitting, the CCFM method simplifies multi-color fluorescence imaging experiments, while keeping a high spatial resolution. However, when the level of the detected fluorescence signal decreases, the image quality, the demosaicking algorithm precision, and the discrimination of fluorescence channels on the colorimetry camera will also decrease. Thus, CCFM has a poor color resolution under a low signal level. For example, the crosstalk will be higher than 10% when the signal is less than 100 photons/pixel. To solve this problem, we developed a new algorithm that combines sCMOS noise correction with demosaicking, and a dye selection method based on the spectral response characteristics of the colorimetry camera. By combining the above two strategies, low crosstalk can be obtained with 4 ∼ 6 fold fewer fluorescence photons, and low light single-frame four-color fluorescence imaging was successfully performed on fixed cos-7 cells. This study expands the power of the CCFM method, and provides a simple and efficient way for various bioimaging applications in low-light conditions.