Ecological processes below ground have the least understood factors in ecosystem structure, functions, and processes. Due to technical and methodological limitations, the underground ecosystem ecology, as a "black box", has become a bottleneck in the development of modern ecology. With the recent development of new DNA methods, the underground ecological research is becoming a hot topic in ecology. Environmental DNA technology includes extraction of DNA fragments directly from environmental samples such as soil, qualitative and quantitative analyses of target organisms by DNA sequencing, and determination of the distribution and functional characteristics of target organisms in the ecosystem. The technology has been successfully applied to study underground ecological processes. Currently, environmental DNA technology is relatively established in the study of microbial diversity and function, and overcomes the long and unsolved issue that most soil microorganisms cannot be cultured. The environmental DNA technology can effectively analyze soil microbial community composition, diversity, and spatial distribution. Microbial function in underground ecological processes can be determined by metagenomics technology. This technology has also been applied to study soil animals; it can quickly analyze diversity and distribution of soil animals, more effectively identify unknown or rare species, and widen the magnitude of the soil fauna identification. Some studies on plant biodiversity and plant taxonomy have utilized extraction and analysis of DNA fragment information in soil. The results obtained by this technology have been more accurate than those obtained by traditional methods. The environmental DNA technology has changed our knowledge about the pattern of plant species diversity. Meanwhile, this technology can overcome the limitations in traditional studies of root systems by reducing the error in root identification, and it can be applied to analyze complex roots involving several species. The environmental DNA technology has also been used to estimate the fine root biomass. In this paper, we summarized the DNA-based molecular methods applied in the study of microbial diversity and function, soil animal diversity, underground plant diversity, roots ecology, and other underground ecological processes, and we believe that our results will provide new ideas and inspiration for studies on complex underground ecology. Environmental DNA technology will be revolutionary in identifying the underground ecosystem structure and function, especially in the ecological processes related to soil microorganisms, animals, plant diversity, and plant roots studies. Future work should focus on three research aspects: (1) ecological function of soil organisms, especially soil animals, by metagenomics technology; (2) application of environmental DNA technology in roots ecology, such as fine root biomass estimation, underground configuration of plant communities, distinguishing roots and rhizosphere, nutrient competition between plants and microbes in soil; and (3) scaling up, from small-scale observations and experiments to the prediction of large-scale patterns as a key to unravel the questions in molecular ecology. It is necessary to fully combine environmental DNA technology with the theory and methods of traditional ecology, especially in the studies of community ecology and ecosystem ecology. Environmental DNA technology has very broad application prospects in underground ecological research. Molecular biology techniques combined with traditional ecological methods will be the future trend of underground ecology.