The present disclosure relates to a method for multi-color fluorescence imaging under a single exposure, an imaging method and system. The imaging system includes: a fluorescence microscope, configured to obtain a real image of the sample; a spatial mask, disposed behind the fluorescence microscope, and configured to perform mask modulation on the real image of the sample; a 4f system, disposed behind the spatial mask, in which the real image of the sample passes through the spatial mask to the 4f system; an optical granting, disposed on a Fourier plane in middle of the 4f system, and configured to split the real image of the sample to obtain a split real image; and an image sensor, configured to obtain the split real image to obtain an image of the sample. The present disclosure advantages of improving imaging rate in multi-spectrum fluorescence microscopy.