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Abstract: The human body is colonized by densely-populated and structurally complex communities of microorganisms. The microbiota interact not only with their host cells, but also with other microbiota. Dual RNA sequencing (Dual RNA-seq) can be used to conduct simultaneous analysis of the dynamic changes of gene expression of two (or more) interactive species, and to obtain thus, through the interaction model diagram, the inter-species regulatory relationship of genes of different species, and hence the interaction mechanism between species. We herein reviewed the application status and development prospects of Dual RNA-seq in the research of intestinal, respiratory, skin and oral microbes. Since the concept of Dual RNA-seq was first introduced, the technology has been applied to a range of infection models. Direct investigation into the dynamic interactions between species at the molecular level will contribute to the better understanding of the physiological changes of pathogens and hosts during the course of infection, and thus help reveal potential new targets or biomarkers. However, the Dual RNA-seq technology is still in its early stage of development, and there are some limitations in the experimental technology. For example, due to the dynamic nature of the interaction between species, there are urgent problems awaiting solutions, such as the optimal experimental conditions, the selection of sampling sites and how to achieve real-time observation. In addition, due to the large amount of bioinformatics data of Dual RNA-seq, further research is needed to explore for ways to process the interaction information quickly and flexibly.