Microbial total DNA obtained from environmental samples comes from cell-free DNA, intracellular DNA of viable and nonviable microbes.The total DNA derived from signals does not directly reflect the structure and composition of microorganisms, and often leads to overestimate the viable microbial population and diversity, thus an accurate and feasible method is urgently required to be developed for screening viable microbes from complex environmental matrices.Propidium monoazide (PMA) is a light activation of DNA bound dye, which selectively permeates only into dead cells with compromised membrane integrity.Upon intercalation in the extracellular DNA and DNA from dead cells (collectively referred as relic DNA), relic DNA is irreversibly modified and inhibited for its PCR amplification.Sample pretreatment with PMA dye combined with quantitative PCR (qPCR) and high-throughput sequencing provides a powerful tool for fast and effectively screening environmental viable microbes.This paper reviewed the recent developments in understanding the detection of environmental viable microbes in sediments, soils and waters based on PMA dye, discussed the current problems, and proposed the strategies of PMA dye application; which would stimulate new ideas for future study of viable microbial communities and functional structures in complex environmental samples.