Traditional microbial identification and cluster analysis method is founded on the basis of the separation of microbial pure culture. Because more than 99% of micro-organisms can not be cultured in the natural environment, microbial analysis and research have many limitations. Compared with the traditional medium-based microbial separation technology as well as physiological methods and molecular biological methods, the Phospholipid Fatty Acid analysis which based on fatty acids as a biomarker is widely used in the research of environmental microbiology nowadays. This paper discusses the PLFA fingerprint analysis and its applications for environmental microbial studies, including identification of unknown microbes and assessment of microbial diversity.