Dolomite is a common carbonate mineral in various sedimentary/diagenetic settings, but its origin has puzzled geologists for decades. Lacustrine dolomite in the lake-marginal environments of rifted basins could have undergone complicated and multiple diagenetic alterations after deposition due to frequent subaerial exposure, faulting, and hydrothermal activity. However, the lack of obvious evidence of fluid activity traces and the small sizes of associated mineral crystals constrain our full understanding of fluid activity in lacustrine dolomite. This study investigated the origin of dolomite and the evolution of diagenetic fluids from the Shahejie Formation in the Huanghekou sag, Bohai Bay Basin, China. The dolomite comprises dolomicrite with minor isopachous dolomite cement and minor Fe-rich saddle dolomite. During the penecontemporaneous period, microbes, including cyanobacteria, Cladosiphonia (green algae), and sulfate-reducing bacteria, may have catalyzed protodolomite precipitation, forming protodolomite associated with Mg-calcite. Later, the protodolomite was transformed into dolomicrite through recrystallization in the early diagenetic stage. Intense evaporation contributed to water-level fluctuations in the shallow lake, and then the metastable protodolomite in the lake-marginal settings was exposed to meteoric water and dissolved. The fluid became oversaturated following extreme capillary evaporation, and isopachous dolomite cement precipitated. During the middle diagenetic period, Fe-rich saddle dolomite precipitated with thermochemical sulfate reduction under the influence of hydrothermal fluid. Well-preserved morphological and distinct geochemical characteristics in this study provide a potential analogue for the origin of ancient lacustrine dolomite in similar sedimentary environments. In addition, a relatively complete fluid evolution history recorded in the dolomite provides important guidance for petroleum exploration in Eocene lacustrine dolomite in the Bohai Bay Basin.