Abstract: The microsporogenesis in Keteleeria fortunei (Murr.) Carr. was investigated using laser scanning confocal microtechnic. Following Eosin/Hoechst33342 double-staining and methyl salicylate clearing, the samples were excited respectively by UV and 488 nm laser to take single scanning images and serial scanning images with a Leica TCS SP2 laser scanning confocal microscope（LSCM）. The LSCM images were then reconstructed with the Leica Confocal Software. The results showed that LSCM optical sections and high contrast images provided clear information about the nuclear developmental stage of microspore, which had previously been difficult to determine using conventional microscopy. During pachytene stage of meiosisⅠof the pollen mother cell（PMC）, the chromosomes compressed and twisted together into a dough, taking up a part of the nuclear with a nucleolus leaning against the dough. There was a diffuse diplotene stage during meiosisⅠof PMC . Cytokinesis at meiosis of PMC was simultaneous. With the staining procedure, spindle at metaphase I 、Ⅱ and callose at tetrad could also be detected. These results also suggested that the Eosin/Hoechst 33342 double-staining technique could be used to highlight the developmental stage of pollen and the LSCM was an ideal tool to study the pollen biology. The above study provided not only the scientific basic for exploring the endangerment mechanism of Keteleeria fortunei, but also significant embryological evidences for the conservation biology and the systematic biology of Keteleeria.