The activated sludge or microbial biofilm are the main habitats of microbial consortia in processes of biological wastewater treatment. The transformation and degradation of pollutants in wastewater can be realized through different metabolic pathways of microorganisms. The microbial community structure directly affects the rate of biotransformation of pollutants and the types of end products, while a comprehensive understanding of the microbial community structure and function can provide a microbiological basis for directional regulation of biological wastewater treatment. Since most of microorganisms are still uncultivable, it becomes a major challenge to reveal microbial dark matter in biological treatment systems. The rapid development of nucleic acid sequencing technique and bioinformatics has promoted the study of environmental microbiology and microbial ecology. In recent years, various meta-omics techniques based on high-throughput sequencing have served as an important tool for studying uncultured microorganisms and unknown genetic resources. Metagenomics and metatranscriptomics can systematically investigate the physiological and metabolic characteristics of microorganisms in specific environments, and provide a better understanding of microbial response and metabolic regulation to environmental variations. Currently, the meta-omics research on microbial dark matter has obtained some novel mechanisms in circulation of substances that break through the traditional understanding. This paper reviews the development of nucleic acid sequencing technique and discusses the recent advances in microbiological researches of biological nitrogen removal, enhanced biological phosphorus removal, and microbial electrochemical techniques based on metagenomics and metatranscriptomics. Finally, we provide an outlook on the prospective development and major challenge for meta-omics researches on biological wastewater treatment.