Revealing the spatio-temporal change of the supply, demand and balance of ecosystem services (ES) associated with human activities and land use changes is of great significance for watershed ecosystem management. Taking the Lancang River Valley as a case, we explicitly studied the ES spatial characteristics using the land use/land cover (LULC) matrix model, Optimized Hot Spot Analysis and landscape pattern analysis. Further, we screened out the dominant explanatory variables which had significant influence on ES supply, demand and balance by means of Geographical Weighted Regression (GWR) method at pixel scale. The results showed that the ES demand intensity varied little throughout the watershed while the downstream ES supply capacity and balance value were greater than upstream. Meanwhile, the hotspots of ES supply and demand were mainly distributed in the south part but coldspots in the north part. Human activity factors integrating landscape pattern variables were verified to have negative impact on ES balance in general. Among them, the Largest Patch Index (LPI) had a negative influence of majority pixels while GDP, cultivated land ratio and Area Weighted Average Patch Fractal Dimension (AWAPFD) had positive effects on a few pixels. This study will provide scientific support for regional ecosystem service trade-off and regulation at multiple scales.