Due to the advanced development of fluorogenic chemistry, quantitative real-time polymerase chain reaction (qRT-PCR) has become an emerging technique for the detection and quantification of microorganisms in the environment. Compared with the conventional hybridization- and PCR-based techniques, qRT-PCR not only has better sensitivity and reproducibility, but it is also quicker to perform and has a minimum risk of amplicon carryover contamination. In this presentation, the principle of this emerging technique, as well as its detection reagents, target DNAs, quantification procedures, and affecting factors will be addressed, followed by a few applications of this techniques in environmental studies, including quantification of two co-culture bacteria in AOC measurement, determination of the functional gene (hydrogenase gene) in a hydrogen-producing microbial community, probing the dominant thermophilic population in an anaerobic phenol-degrading sludge, and measurement of the rrn operon copy number of bacteria.