The micromorphology and charge characteristics of river sediments play a crucial role in their ability to adsorb dissolved nutrients and pollutants, serving as essential carriers of biogenic elements in aquatic ecosystems. This study focused on the mainstream and major tributaries in the middle and lower reaches of the Lancang River. We collected suspended sediments from 28 river sections and utilized atomic force microscopy to measure their microsurface charges, aiming to illuminate the microsurface charge properties of Lancang River sediments and, by extension, the microsurface properties of bottom sediment. Our research revealed key findings: (1) Sediment morphology significantly influenced surface charge distribution at the microscopic level. Potential and phase difference maps showed distinct non-uniform patterns at the nanoscale. (2) Substantial variations in surface charge existed across different watershed regions, with changes along the watershed having a pronounced impact. These changes followed a decreasing-then-increasing trend, displaying surface potential values from -201.47 to 35.34 mV and surface charge density varying from 0.07 to 3.65 mC/m². (3) The construction of terraced hydropower dams notably influenced sediment surface charge. The potential difference between sediment particles upstream and downstream of the dam exhibited a significant linear correlation with reservoir capacity, with a correlation coefficient of 0.8214; (4) The surface potential of sediment demonstrated a robust correlation with the variation pattern of phosphorus, as evidenced by a correlation coefficient of 0.6657. This relationship was further underscored by a well-fitted linear model. Generally, potentials were higher below the dam compared to above the dam. These research findings have significant scientific implications for understanding pollutant transport within watersheds, elucidating the surface electrical attributes of sediment, and comprehending the mechanisms of pollutant adsorption and desorption.