Arbuscular mycorrhizal (AM) fungi are one of the most important soil microorganisms in terrestrial ecosystems. The arbuscular mycorrhizal network is formed by extraradical mycelium of AM fungi that connect two or more plant roots within the soil system. How to discover the roles of AM fungi in soil ecosystems has been a major focus of ecological research. It has been found that isotope tracer technique could be used for research on AM fungi and its role in nutrient absorption and transportation from soil to host plants. The isotope tracer technique could also be used to identify the mycorrhizal network distribution between different host plants, and to study the role of AM fungi in the ecosystem. To illustrate the value of the isotope tracer technique in AM fungi research, our study combined the latest research findings in the field of mycorrhizal ecology with the present findings and focused on: 1) the application of the isotope tracer technique in exploring the mechanisms of absorption and transportation of different elements in AM symbionts; 2) the value of the isotope tracer technique in arbuscular mycorrhizal network studies; 3) the use of the isotope tracer technique to study the roles of AM fungi in the ecosystem. Thus, the aim of this review was to provide a theoretical basis for, and to predict future prospects of the use of the isotope tracer technique in AM fungi research.