The invention relates to the field of microorganisms, and particularly relates to a preparation method and application of a pair of double-nitrogen-containing alkaloid enantiomers derived from deep-sea sediment fungi. The specific structural formula of the enantiomers is shown as formula (I). The preparation method comprises the following steps : inoculating a fungus medium with fungi Penicillium oxalicum 13-37 derived from deep-sea sediment for fermentation culture, and separating and purifying the fermentation product to obtain a pair of double-nitrogen-containing alkaloid enantiomers (R) and(S) shown as formula (I). The pair of double-nitrogen-containing alkaloid enantiomers obtained by the invention has an inhibitory effect on tumor cells, and the compound also has bacteriostatic activity.
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