Dolomite genesis is a century-old mystery in sedimentology. To reveal the mechanism of dolomite genesis, two core problems need to be addressed. The first is the origin and migration mechanism of Mg2+-rich fluids during the dolomitization process. The second is the kinetic barrier caused by Mg2+ hydration during dolomite precipitation at low temperatures. To address these problems, our study, based on detailed petrological, sedimentological, geochemical (major and trace elements), and isotopic (C-O-Mg) analysis, clarified the source and migration of Mg2+-rich fluids and the kinetic barrier mechanism of low-temperature dolomite precipitation in the Upper Sinian Qigebulake Formation and the Lower Cambrian Xiaoerbulake Formation in the Tarim Basin. First, we found that the Mg2+-rich fluids required for the dolomitization of dolomite in the Xiaoerbulake Formation were primarily derived from the Early Cambrian marine fluid. At the interface of the sedimentary cycle, δ 26Mg values fluctuated considerably, indicating that the sequence interface was the starting point and channel for the migration of dolomitized fluids. Sea level variation plays a major role in controlling the dolomitization process of the Xiaoerbulake Formation. Second, the Qigebulake Formation contains low-temperature dolomite with Mg2+-rich fluids supplied by seawater, microorganisms, and sedimentary organic matter. Comprehensive analysis shows that the dolomite of the Qigebulake Formation was formed by microbial induction by anaerobic methane bacteria. Finally, the properties and sources of dolomitization fluids and the formation process of dolomite were the reasons for the difference in the Mg isotope composition of dolomite during the Sinian-Cambrian transition. This study reveals the genetic mechanism of the Sinian-Cambrian dolomite in the Tarim Basin and establishes a new method to explain the genesis of microbial dolomite by C-O-Mg isotopes, providing a reference for the reconstruction of the formation and evolution of dolomites.