In order to prepare fumitremorgin B (FTB), strains of Aspergillus fumigatus C4104 and 3656 were selected, based on the screening of high-FTB-production strains, and inoculated in rice medium of five kilograms and incubated for producing toxin. Cultures were extracted with ethyl acetate, dehydrated with anhydrous sodium sulfate, defatted with n-hexane, decolorized with activated charcoal, chromatographed in silica gel H column, recrystallized by methanol, and finally, four grams of colorless fine-needle shaped crystal were harvested. Two strains of the fungus could produce 800 mg of toxin per kilogram rice medium in average with a yield of approximately 20%, much higher than that reported in literatures (1.11%). Indentification with chemical analysis, such as melting point, ultraviolet, infrared, elemental analysis, mass spectrometry, as well as analyses of nuclear magnetic resonance hydrogen spectrum and carbon spectrum, showed consistency of the results with those in literatures, and confirmed it contained FTB with one molecule of H2O. Contribution of carbon and hydrogen to the molecular structure of self-prepared FTB was explored in the paper based on the spectrometric data.