Sediment is an important place for the circulation of nitrogen and phosphorus in river ecosystems, and microorganisms are an important part of river ecosystems. It is of great significance to explore the carbon source utilization characteristics and functional diversity of microbial communities in sediments for river ecological environmental protection. In this study, the Biolog Eco microplate method, principal component analysis and redundancy analysis were used to elucidate the variation characteristics and influencing factors of carbon source utilization intensity and functional diversity of microbial communities in the sediments of the Dali River Basin. The results show that:(1) from the upstream to the downstream of the watershed, the utilization intensity of microbial carbon sources in the sediments gradually decrease. Compare with the upstream, the utilization intensity of microbial carbon sources in the tributaries, midstream and downstream sediments decrease by 13.4%, 30.5%, and 30.7%. (2) There are differences in the functional diversity of microbial communities in sediments. The functional diversity of microbial communities in sediments (Shannon-Wiener diversity index) is upstream > tributaries > midstream > downstream, and common species dominance (Simpson diversity index) is shown as downstream > tributary > midstream > upstream. (3) Carbohydrates are highly correlated with microbial metabolic activity; amino acids, polymers, carboxylic acids, amines, and phenolic acids are less correlated with microbial metabolic activity. (4) The contents of total nitrogen, ammonia nitrogen, nitrate nitrogen and organic carbon in the sediments are the main factors affecting the functional diversity of microbial communities and differences in carbon source utilization characteristics. Appropriate carbon and nitrogen levels in river basin sediments are of great significance to maintain the ecological health of river water.