Abstract: iming at solving the difficulty of extracting lipids from Bacillus spores by traditional Bligh & Dyer method, Geobacillus stearothermophilus ( G. stearothermophilus ) is investigated to analyze extraction of spores' lipids and construction of its profile. The results show that spore layers, from the outside , including spore coat, outer membrane and peptidoglycan can be stripped by alkaline denaturing reagents and lysozyme, exposing inner membrane rich in lipids on the naked spore. Fluorescence staining further confirms that the spore layers are efficiently stripped. Lipids in the naked spores are extracted and analyzed by using MALDI-TOF-MS. The lipidomic study shows that the spores are mainly consisted of phosphatidylglycerol, phosphoethanolamine and its derivatives, and cardiolipins.