The fluorescent green spot disease is a physiological disease of tea mature leaves. The green spots on the leaf back emit yellow-greenish fluorescence. In order to investigate the rule of the disease occurrence by using spontaneous fluorescence as a probe and the spectra of yellow-greenish fluorescent compounds for their isolation and identification, the fluorescence microscopy, microscopic fluorescence spectral imaging, laser scanning confocal microscopy and flow cytometry were used in this investigation. This experiment covered the condition of microscopic observation, emission spectral characters of yellow-greenish spontaneous fluorescence and their localization in the diseased tea leaves. Results showed that the diseased leaves could emit several kinds of spontaneous fluorescence by irradiating different lights, and the optimal microscopic condition for observing the yellow-greenish fluorescence was that the diseased leaves were excited by blue light and images recorded in multicolor and green color mode, in which there were three fluorescence peaks at 515 nm, 535 nm, and 585 nm within the light range of yellow and green. The yellow-greenish fluorescence were firstly emitted in the sheath cell of vascular bundle and then in the barrier cells, sponge cells and the epidermis cells. And the location of fluorescence was the vacuole of the diseased cells.