The developing of modern molecular biology have been widely applied in the development of novel technologies and approaches used in the studies of microbial diversity, which have been accelerating the formation of new research fields such as modern molecular microbial ecology and its related subjects. The technology of Metagenomic Clone Library Analysis is one of such technologies recently used in the study of microbial ecology. In this review, we systematically reviewed and discussed the backgrounds of formation, principle and applications of this technology in the microbial ecological studies. Furthermore, the applications of this method in the discovery of new gene and novel biocatalysts, in the study of human metagenomics and microbial biodiversity in a community were extensively discussed here. We also summarized different approaches currently used in screenings of metagenomic clone library including PCR-based screening, fluorescent in situ hybridization (FISH), substrate induced gene expression screening (SIGEX) and microarray. The advantages and disadvantages of above screening approaches were compared and the future direction of development of this technology was also discussed in this article.