Environmental transcriptomics, which focuses on microbial mRNA derived from complex environmental samples using the RNA-Seq method, allows investigation of expression and patterns of regulation of functional genes in natural microbial communities. This review outlines the basic protocol of environmental transcriptomics, from sample collection and preservation, total RNA isolation, mRNA enrichment, cDNA synthesis to high-throughput sequencing and data analysis. Main technological problems are pointed out, such as low yield of mRNA in environmental samples, contamination of mRNA by various impurities like humic substances and limited degree of rRNA removal. Recent progresses in specific methodologies to improve the quantity and quality of mRNA, especially in RNA extraction, purification and the enrichment of mRNA, are outlined. Bioinformatics methods that deal with the large volume of RNA-Seq data are addressed, such as quality control of the sequence data, sequence assembly, detection and removal of rRNA, gene annotation and functional classification, and detection of differently expressed genes. The widely application of environmental transcriptomics, including detection of new genes, study of gene expression and regulation of microorganisms in different environments, and the analysis of metabolic pathways of special organic substances, are also highlighted. Environmental transcriptomics, combined with the further development of sequencing technology and bioinformatics tools in the future, are likely to be comprehensively used in the study of environmental microbiology.