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The present invention relates to a method for preparing a genomic library derived from a single cell of rare microorganisms or novel microorganisms. More specifically, the present invention is characterized by : analyzing a community of a microorganism group from collected samples; performing FISH and first FACS process for removing a dominant microorganism group based on the analysis and performing second FACS process to sort the microorganisms into single cells; amplifying non-culturable rare microorganisms or novel microorganisms which rarely exist in an environment into a genome derived from a single cell from performing multiple displacement amplification; and constructing a gene library. The method of the prevent invention can obtain and provide a genome library of non-culturable microorganisms and novel microorganisms existing in a real environment, which are difficult to detect by a conventional method and can obtain large quantities of genes of rare and novel microorganisms. In addition, the method is useful since the method enables the identification of the origin of the obtained gene unlike the conventional method for preparing a metagenome library and a library based on diversity can be manufactured by removing a dominant microorganism existing in an environment.