Efficient photosynthesis reaction thanks to the flexible energy regulation of two important pigment-protein complexes 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 insufficiency 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. Development to date, the combination of optical microscopies with various spectroscopic techniques has extended the possibility of a multi-perspective investigation in photosynthesis research. In this paper, I review the important and recent progress in cryogenic spectral microscopy in the field of natural photosynthesis research from two aspects: single-molecule spectroscopy and single-cell spectroscopy. Meanwhile, I illustrate the advantages of this technique in clarifying the correlation between structure variability and function of pigment-protein complexes, and the physiological responses of photosynthetic organisms to variable environments.