Assessing the scale effects of land use on water quality is of great significance for effectively controlling nonpoint source (NPS) pollution in river basins. In this study, redundancy analysis (RDA) and stepwise multiple linear regression (SMLR) analysis were applied to assess the effects of land use on water quality across multiscales in the Tuojiang River Basin. All monitoring sections were classified into three groups according to the characteristics of land use and cluster analysis of water quality. Results showed that the improvement in water quality of rivers in the Tuojiang River Basin lies in the emphasis and protection of the small-scale scope. Concomitantly, the linkages between individual water quality parameter and land use were highly dependent on spatial scales and regional basis. For the upstream group A, urban land is the main source of COD and TN pollution, while industrial and rural residential land contributed the most to TP pollution. Water body exhibits favorable effects on ammonia nitrogen due to its absorption and degradation, together with the growth of phytoplankton within it. For group B in the middle-lower reaches, controlling the input of organic fertilizers in paddy field will effectively alleviate COD pollution. Increasing the proportion of grassland near the riparian zone can have a positive effect on TN and TP pollution. It should continue to strengthen the strict supervision of NH3-N concentration in wastewater discharge from industrial enterprises. Our results can provide important information for land use planning and making multiple scale measures for water quality conservation.