The invention provides a multi-band spectral imaging light source, an imaging method and a microscope. The multi-band spectral imaging light source comprises a first combined light source, a first collimator, a mirror, a first semipermeable half mirror, a second combined light source and a second collimator. The first combined light source is capable of emitting at least one kind of light of a monochromatic wavelength, and the light is collimated by the first collimator and comes to the mirror face of the mirror at a first incident angle. The first semipermeable half mirror is arranged on a first light path between the first combined light source and the mirror. The second combined light source is arranged at a position other than the first light path. The multi-band spectral imaging light source and the imaging method have the advantages of low cost and rapid imaging. By using the method and the light source, multi-band multi-spectral imaging above 60 frames/second can be realized, which satisfies the requirements of most of the medical multi-spectral imaging applications.