Efficient photosynthesis reaction is attributed to the flexible energy regulation of two important pigment-protein complexes, i.e. photosystem II (PSII) and photosystem I (PSI). Cryogenic spectral microscopy provides information about the spatial distribution and physiological functional states of photosynthetic components in photosynthetic organisms. Under low temperatures, the uphill energy transfer between pigments is efficiently suppressed so that the temperature-dependent PSI can be well analyzed. Therefore, a cryogenic spectral microscope allows us to discuss the physiological events surrounding PSII and PSI in the independent microscopic zones. This technique can be used to complement the insufficiencies of cryogenic electron microscopy and atomic force microscopy in analyzing the photophysics and photochemistry of photosynthetic species. Historically, cryogenic optical microscopes originated from the desire for single-molecule spectroscopy detection. So far, the combination of optical microscopies and various spectroscopic techniques has expanded the possibility of studying photosynthesis from multiple perspectives. In this paper, the important and recent progress of cryogenic spectral microscopy in the field of natural photosynthesis research is reviewed from two aspects: single-molecule spectroscopy and single-cell spectroscopy, and the advantages of this technique in clarifying the correlation between structure variability and function of pigment-protein complexes, as well as the physiological responses of photosynthetic organisms to variable environments, are also illustrated.