Microbial metagenome is the largest gene reservoir in environment. Exploring uncultured microbial gene resource has been restricted, due to the fact that only 0.1%-1% of the microorganisms can be cultivated using current techniques. Metagenome libraries can be constructed by direct extracting DNA from environmental samples to avoid the limitations of culture-dependent methods. Metagenomic technique has greatly enhanced the utilization of microbial resource with more opportunity of obtaining novel bioactive compounds. In this paper, the concept of metagenome, basic operation process and some key technical details are introduced. The introduction is focused on the advancement in some “bottle neck” techniques, including enrichment of interested genes, extraction of nucleic acid, selection of vector and host system, and metagenomic library screening. Gene enrichment involves techniques such as Stable Isotope Probing (SIP), Suppression Subtractive Hybridization (SSH), and Differential Display (DD). Metagenomic library screening includes sequence-dependent and sequence-independent method. The former method includes gene-specific Polymerase Chain Reaction (PCR), reverse transcription PCR (RT-PCR), DNA microarray, affinity capture, and so on. The latter method is mainly referred as activity-based screening and “gene trap” techniques. Some recent examples of gene targeting through metagenome libraries are also introduced.