Abstract: Exploring the trade-offs and synergies among various ecosystem services (ESs) in river basins is crucial for providing scientific references for comprehensive ecological governance and promoting the sustainable development of ecosystems. As a key ecological and economic region in eastern China, the Qiantang River Basin has undergone significant environmental changes amid rapid urbanization and land use transformation over the past two decades. To systematically understand the spatio-temporal dynamics of ESs and their internal relationships in this basin, this study employed the InVEST (Integrated Valuation of Ecosystem Services and Trade-offs) model and spatial analysis techniques to investigate the spatio-temporal evolution of land use types, quantify the trade-off and synergy relationships among typical ESs, and delineate the spatial distribution of ecosystem service bundles (ESBs) in the Qiantang River Basin from 2000 to 2020. The core ESs evaluated included carbon storage (CS), habitat quality (HQ), soil conservation (SC), water yield (WY), and water conservation supply (WC), with their relationships analyzed using correlation analysis and spatial clustering methods. The results are as follows: (1) The area of construction land in the basin increased by 757.16 km² during the study period, while forest land and cultivated land decreased by 296.70 and 3 429.14 km², respectively. This land use conversion primarily reflected the expansion of urban and built-up areas driven by socio-economic development. (2) Temporally, CS and HQ showed a continuous declining trend over the 20-year period, whereas SC, WY, and WC exhibited a pattern of first increasing and then decreasing, with their peak values occurring around the mid-study period. (3) Synergistic relationships dominated among the assessed ESs: significant positive synergies were observed between CS and HQ, CS and WC, and HQ and WC, indicating that the improvement of one service often promoted the enhancement of the others. (4) Spatially, the ESBs in the basin were mainly dominated by transitional ecological conservation bundles and core ecosystem service bundles, which accounted for 48.28% and 36.81% of the total basin area on average over the 20-year study period, respectively. The spatio-temporal distribution of these ESBs showed high consistency with land use types, reflecting the strong influence of land use patterns on ES aggregation. (5) Vegetation coverage and topographic factors were the primary driving forces affecting the spatio-temporal differentiation of CS and HQ in the basin, while precipitation and topographic differences played important roles in shaping the spatio-temporal variations of SC, WC, and WY. This study provides a comprehensive understanding of the dynamics of ESs and their underlying mechanisms in the Qiantang River Basin. The findings offer valuable scientific references for the comprehensive ecological governance of the Qiantang River Basin and other similar river basins globally, and they support the formulation of targeted policies to balance ecological protection and socio-economic development.