The invention is applicable to the field of optics, biomedicine, life science and the like, and provides a two channel-based multi-spectrum fluorescent imaging microscopic system and method, wherein the two channel-based multi-spectrum fluorescent imaging microscopic system comprises a picosecond pulse laser device, a fluorescent excitation and collection light path, a microscopic objective lens, a light beam lens, a double-ICCD detector, and a control and processing module. The invention further discloses a method for performing multi-spectrum imaging by utilizing the two channel-based multi-spectrum fluorescent imaging microscopic system. According to the two channel-based multi-spectrum fluorescent imaging microscopic system and method, the limitation of the existing fluorescent microscope and a fluorescent life imaging microscopic system only can acquire single wavelength fluorescent signal with one-time detection can be effectively solved, the simultaneous acquisition of the multi-spectrum fluorescent strength and fluorescent light image aiming at the dynamic process of fluorescent intensity-related detection limited in biomedicine and life science can be performed, so that the research and application ranges of biophotonics can be extended.