It has evolved as a main limiting factor for the exploitation and application of microbial resources that more than 99% of microorganisms in natural environments cannot be cultivated so far by current isolation and culture methods. Metagenomics is an advanced methodology by means of extracting all microbial genomic DNAs in certain environmental habitat, constructing and screening metagenomic libraries to seek novel functional genes and/or biologically active compounds. It can overcome the advantages of isolation and cultivation procedures in traditional microbial method, and thus greatly broaden the space of microbial resource utilization. So it has become one of the powerful research tools for microbiology, biotechnology, soil and environmental sciences, and a new field of genetic engineering. This paper mainly reviewed the metagenomic methodology including genomic DNA isolation, library construction and screening strategies. And this review also outlined the up-to-date applications of metagenomics in various fields.